Cas9 fusion molecules, gene editing systems, and methods of use thereof

ABSTRACT

Disclosed herein are enzymatically active Cas9 (eaCas9) fusion molecules, comprising an eaCas9 molecule linked, e.g., covalently or non-covalently, to a template nucleic acid; gene editing systems comprising the eaCas9 fusion molecules, and methods of use thereof.

RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. _(16/093,336), filed on Oct. 12, 2018; which is a35 U.S.C. § 371 national stage filing of International Application No.PCT/US2017/027126, filed on Apr. 12, 2017; which claims priority to U.S.Provisional Application No. 62/322,026, filed on Apr. 13, 2016. Theentire contents of each of the aforementioned applications are expresslyincorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Apr. 11, 2017, isnamed EM057US2_SL_2017-04-12.txt and is 257,840 bytes in size.

FIELD OF THE INVENTION

The invention relates to Cas9 fusion molecules and methods andcomponents for increasing editing of a target nucleic acid sequence bygene correction using an exogenous homologous region, and applicationsthereof.

BACKGROUND

The CRISPR (Clustered Regularly Interspaced Short PalindromicRepeats)/Cas (CRISPR-associated) system evolved in bacteria and archaeaas an adaptive immune system to defend against viral attack. Uponexposure to a virus, short segments of viral DNA are integrated into theCRISPR locus. RNA is transcribed from a portion of the CRISPR locus thatincludes the viral sequence. That RNA, which contains a sequencecomplimentary to the viral genome, mediates targeting of a Cas9 proteinto the sequence in the viral genome. The Cas9 protein cleaves andthereby silences the viral target.

Recently, the CRISPR/Cas system has attracted widespread interest as atool for genome editing through the generation of site-specific doublestrand breaks (DSBs). Current CRISPR/Cas systems that generatesite-specific DSBs can be used to edit DNA in eukaryotic cells, e.g., byproducing deletions, insertions and/or changes in nucleotide sequence.

Without wishing to be bound by any theory, it is thought that themechanism by which an individual DSB is repaired varies depending onwhether or not the DNA ends created by the DSB undergo endo- orexonucleolytic processing (also referred to as “end resection” or“processing”). When no end resection takes place, a DSB is generallyrepaired by a pathway referred to as classical non-homologous endjoining (C-NHEJ). C-NHEJ is considered an “error-prone” pathway inasmuchas it leads in some cases to the formation of small insertions anddeletions, though it may also result in perfect repair of DSBs withoutsequence alterations.

In contrast, if end resection does take place, the ends of a DSB mayinclude one or more overhangs (for example, 3′ overhangs or 5′overhangs), which can interact with nearby homologous sequences. Again,the mechanism by which the DSB is repaired may vary depending on theextent of processing. When the ends of a DSB undergo relatively limitedend resection, the DSB is generally processed by alternativenon-homologous end joining (ALT-NHEJ), a class of pathways that includesblunt end-joining (blunt EJ), microhomology mediated end joining (MMEJ),and synthesis dependent micro homology mediated end joining (SD-MMEJ).However, when end resection is extensive, the resulting overhangs mayundergo strand invasion of highly homologous sequences (which can beendogenous sequences, for instance from a sister chromatid, orheterologous sequences from an exogenous template), followed by repairof the DSB by a homology-dependent recombination (HDR) pathway.

While a cell could, in theory, repair DNA breaks via any of a number ofDNA damage repair pathways, in certain circumstances it is useful ordesirable to manipulate the local environment in which the DSB is formedin order to drive a particular mode of repair. For instance, theaddition of an exogenous homologous DNA sequence (also referred to as a“donor template” or “template nucleic acid”) to a CRISPR/Cas system maytend to drive repair of DSBs through HDR-based gene correction. However,gene correction strategies that rely on exogenous donor templates arecomplicated by the potential for interactions between the donortemplate, the Cas9 and the guide RNA. At the same time, because thedonor template is not a naturally occurring part of the CRISPR/Cascomplex it may only be present and accessible at a fraction of the DSBsformed by the CRISPR/Cas system, and the desired gene correction mayonly occur in a fraction of instances.

SUMMARY

This disclosure provides systems, methods and compositions thatfacilitate gene correction by reconciling the need to localize the donortemplate at DSBs with the need to prevent interactions between the donortemplate and the guide RNA or the Cas9. In the various aspects of thedisclosure, one or more Cas9 fusion molecules comprising a Cas9polypeptide linked to a template nucleic acid sequence are utilized toincrease the frequency and efficiency of DNA repair of DSBs using genecorrection. The Cas9 fusion molecules of the invention comprise Cas9molecules linked both covalently and non-covalently to template nucleicacids. While not wishing to be bound by theory, it is believed that, byoptimizing the length of a linker between the Cas9 polypeptide and thetemplate nucleic acid, hybridization of the template nucleic acid to agRNA associated with the Cas9 molecule, and/or interactions between thetemplate nucleic acid and DNA binding regions of Cas9, are reduced oreven eliminated, while at the same time ensuring that the templatenucleic acid is available to participate in HDR, thereby improving theefficiency of gene correction. In some cases, the efficiency of DNArepair via gene correction pathways may be significantly enhanced (e.g.,doubled) when the donor template is linked to the Cas9 molecule, ascompared to the un-linked molecule. Again, without wishing to be boundby any theory, it is also believed that by linking the donor template tothe Cas9, the potential for degradation of the donor template (e.g.,during trafficking into the nucleus) is reduced and nuclear localizationof the template is improved.

In one aspect, this disclosure relates to compositions and methods formodifying a target nucleic acid in a cell, involving an enzymaticallyactive Cas9 (eaCas9) fusion molecule. For example, the enzymaticallyactive Cas9 (eaCas9) fusion molecule may be an eaCas9 molecule linked toa template nucleic acid.

In one embodiment, the eaCas9 molecule is covalently linked to thetemplate nucleic acid. In certain embodiments, the eaCas9 molecule iscovalently linked to the template nucleic acid using a polypeptidelinker. In some embodiments, the polypeptide linker has a lengthsufficient to reduce or prevent hybridization of the template nucleicacid to a gRNA molecule associated with the eaCas9 molecule. In otherembodiments, the polypeptide linker is sufficiently long to allow theeaCas9 molecule to bind to a target nucleic acid without stericinterference. In other embodiments, the polypeptide linker issufficiently long to allow the template nucleic acid to interact withthe eaCas9 molecule without steric interference.

In certain embodiments, the polypeptide linker is between about 3 andabout 100 amino acids in length. In some embodiments, the polypeptidelinker is an XTEN linker, e.g., an XTEN linker with the amino acidsequence SGSETPGTSESATPES. In other embodiments, the polypeptide linkeris a GGS₉ linker that is 27 amino acids in length. In other embodiments,the polypeptide linker is a GGS₆ linker that is 18 amino acids inlength. In another embodiment, the polypeptide linker is a GGS linkerthat is 3 amino acids in length.

In one embodiment, the eaCas9 molecule is a variant eaCas9 moleculewhich has been modified to have at least one modification at a surfaceexposed residue. In some embodiments, the eaCas9 molecule has at leastone modification a non-cysteine amino acid residue to a cysteine aminoacid residue. In one embodiment, the eaCas9 molecule is a variant eaCas9molecule which has a surface exposed thiol group.

In one embodiment, a template nucleic acid has a maleimide modification.In another embodiment, a variant eaCas9 molecule, for example, an eaCas9molecule which has a surface exposed thiol group, is linked to thetemplate nucleic acid having a maleimide modification using thiolcoupling.

In one embodiment, an eaCas9 molecule which has a surface exposed thiolgroup, is linked to the template nucleic acid having an acryditemodification using thiol coupling.

In one embodiment, an eaCas9 molecule has been modified to have asuccinimidyl-6-hydrazino-nicotinamide modification. In anotherembodiment, the succinimidyl-6-hydrazino-nicotinamide modified eaCas9(i.e., the S-HyNic eaCas9 molecule) molecule is linked a templatenucleic acid having a 4Fb modification, using amine coupling.

In one embodiment, an eaCas9 molecule has been modified to have aHaloTag®, a SNAP-Tag®, a CLIP-Tag®, a ACP-Tag®, or a MCP-Tag® linked tothe eaCas9 molecule. For example, in certain embodiments, aneaCas9-HaloTag molecule is lined to a haloalkane modified templatenucleic acid, e.g., using an S_(N)2 reaction.

In one embodiment, the eaCas9 molecule is covalently linked to thetemplate nucleic acid using a synthetic linker. In certain embodiments,the eaCas9 molecule is a variant eaCas9 molecule which has been modifiedto have a N-[ε-Maleimidocaproic acid] hydrazide (EMCH) and a1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) coupling agent. Inother embodiments, the template nucleic acid has a carboxy group, suchthat the variant eaCas9 molecule is linked to the template nucleic acidby conjugation of the carboxy group on the template nucleic acid to aprimary amine of a hydrazine group on the variant Cas9 molecule whichhas been modified to have a N-[ε-Maleimidocaproic acid] hydrazide (EMCH)and a 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) couplingagent.

In one embodiment, the eaCas9 molecule is non-covalently linked to thetemplate nucleic acid. In certain embodiments, the eaCas9 molecule iscovalently linked to a first ligand, the template nucleic acid iscovalently linked to a second ligand, and the first ligand and thesecond ligand are non-covalently linked to a ligand acceptor molecule.For example, in some embodiments, the first and second ligands arebiotin. In some embodiments, the ligand acceptor molecule isstreptavidin. In another embodiment, eaCas9 molecule non-covalentlylinked to the template nucleic acid, includes a linker between theeaCas9 molecule and the first ligand. In some embodiments, the linker issufficiently long to allow the eaCas9 molecule to bind to a targetnucleic acid. For example, the linker may be sufficiently long to allowthe template nucleic acid to interact with the eaCas9 molecule withoutsteric interference.

In one embodiment, the eaCas9 molecule is covalently linked to apolypeptide, and the polypeptide is non-covalently bound to the templatenucleic acid. In some embodiments, the polypeptide is a nucleic acidbinding protein. For example, the nucleic acid binding protein may beany one of Rad52, Rad52-yeast, RPA-4 subunit, BRCA2, Rad51, Rad51B,Rad51C, XRCC2, XRCC3, RecA, RadA, HNRNPA1, UP1 Filament of HNRNPA1,NABP2 (SSB1), NABP1 (SSB2), and UHIRF1.

In one embodiment, a eaCas9 fusion molecule includes a template nucleicacid having a double stranded nucleic acid sequence or a single strandednucleic acid sequence.

In one embodiment, a eaCas9 fusion molecule includes a wild-type Cas9molecule

In one embodiment, a eaCas9 fusion molecule includes a Cas9 nickasemolecule.

In one embodiment, a eaCas9 fusion molecule includes a split Cas9molecule or an inducible Cas9 molecule.

In one embodiment, a gene editing system includes at least one eaCas9fusion molecule having an eaCas9 molecule linked to a template nucleicacid and at least one gRNA molecule. In some embodiments, the geneediting system, which includes at least one gRNA molecule and a eaCas9fusion molecule, are designed to associate with a target nucleic acidand generate a double strand break on the target nucleic acid. In someembodiments, the double strand break is repaired by at least one DNArepair pathway, thereby producing a modified target nucleic acid.

In one embodiment, a gene editing system includes a first eaCas9 fusionmolecule, which includes a first Cas9 nickase molecule linked to a firsttemplate nucleic acid, a first gRNA molecule, a second eaCas9 fusionmolecule, which includes a second Cas9 nickase molecule linked to asecond template nucleic acid; and a second gRNA molecule.

In one embodiment, a gene editing system includes a first eaCas9 fusionmolecule, which includes a first Cas9 nickase molecule linked to atemplate nucleic acid and a first gRNA molecule.

In one embodiment, a gene editing system includes a first eaCas9 fusionmolecule, which includes a first Cas9 nickase molecule linked to atemplate nucleic acid, a first gRNA molecule, a second eaCas9 fusion,which includes a second Cas9 nickase molecule linked to a templatenucleic acid and a first gRNA molecule.

In one embodiment, a gene editing system includes a first eaCas9 fusionmolecule, which includes a first Cas9 nickase molecule linked to a firsttemplate nucleic acid, a first gRNA molecule, a second eaCas9 fusionmolecule, which includes a second Cas9 nickase molecule linked to asecond template nucleic acid, and a second gRNA molecule. In someembodiments,

the first gRNA molecule and the first eaCas9 fusion molecule aredesigned to associate with a target nucleic acid and generate a firstsingle strand break on a first strand of the target nucleic acid, andthe second gRNA molecule and the second eaCas9 fusion molecule aredesigned to associate with the target nucleic acid and generate a secondsingle strand break on a second strand of the target nucleic acid,forming a double strand break in the target nucleic acid having a firstoverhang and a second overhang, such that the double strand break isrepaired by at least one DNA repair pathway, thereby producing amodified target nucleic acid.

In one embodiment, each Cas9 nickase molecule has N-terminal RuvC-likedomain cleavage activity, but no HNH-like domain cleavage activity. Inother embodiments, each Cas9 nickase molecule comprises an amino acidmutation at an amino acid position corresponding to amino acid positionN863 of Streptococcus pyogenes Cas9.

In one embodiment, each Cas9 nickase molecule has HNH-like domaincleavage activity but no N-terminal RuvC-like domain cleavage activity.In other embodiments, each Cas9 nickase molecule comprises an amino acidmutation at an amino acid position corresponding to amino acid positionD10 of Streptococcus pyogenes Cas9.

In one embodiment, a gene editing system includes an enzymaticallyinactive Cas9 (eiCas9) molecule

In one embodiment, a cell includes a gene editing system as disclosedherein.

In one embodiment, a pharmaceutical composition includes a gene editingsystem as disclosed herein.

In one embodiment, a method of modifying a target nucleic acid in a cellincludes contacting a cell with a gRNA molecule and an eaCas9 fusionmolecule, which includes an eaCas9 molecule linked to a template nucleicacid. In some embodiments, the gRNA molecule and the eaCas9 fusionmolecule associate with the target nucleic acid and generate a doublestrand break in the target nucleic acid, such that the double strandbreak is repaired by gene correction using the template nucleic acid ofthe eaCas9 fusion molecule.

In one embodiment, a method of modifying a target nucleic acid in a cellincludes contacting a cell with a first gRNA molecule, a first eaCas9molecule, a second gRNA molecule; and a second eaCas9 molecule; suchthat at least one of the first and second eaCas9 molecule is linked to atemplate nucleic acid, further such that the first gRNA molecule and thefirst eaCas9 molecule associate with the target nucleic acid andgenerate a first single strand cleavage event on a first strand of thetarget nucleic acid. In some embodiments, the second gRNA molecule andthe second eaCas9 molecule associate with the target nucleic acid andgenerate a second single strand cleavage event on a second strand of thetarget nucleic acid, such that a double strand break having a firstoverhang and a second overhang is formed, and such that the firstoverhang and the second overhang in the target nucleic acid are repairedby gene correction using the template nucleic acid. In one embodiment,the first eaCas9 molecule is linked to the template nucleic acid. Inanother embodiment, both the first eaCas9 molecule and the second eaCas9molecule are linked to the template nucleic acid.

In one embodiment, each eaCas9 has N-terminal RuvC-like domain cleavageactivity, but no HNH-like domain cleavage activity. In anotherembodiment, each eaCas9 has an amino acid mutation at an amino acidposition corresponding to amino acid position N863 of Streptococcuspyogenes Cas9.

In one embodiment, each eaCas9 has HNH-like domain cleavage activity butno N-terminal RuvC-like domain cleavage activity. In another embodiment,each eaCas9 has an amino acid mutation at an amino acid positioncorresponding to amino acid position D10 of Streptococcus pyogenes Cas9.

In one embodiment, the cell is a mammalian cell. In other embodiments,the cell is a human cell.

In one embodiment, a cell is altered by any of the methods disclosed ordescribed herein.

In one embodiment, a pharmaceutical composition includes a cell alteredby any of the methods disclosed or described herein.

In one embodiment, a nucleic acid molecule encodes at least one eaCas9fusion molecule, which includes an eaCas9 molecule and a polypeptide. Insome embodiments, the polypeptide is a nucleic acid binding protein. Insome embodiments, the nucleic acid binding protein may be any one ofRad52, Rad52-yeast, RPA-4 subunit, BRCA2, Rad51, Rad51B, Rad51C, XRCC2,XRCC3, RecA, RadA, HNRNPA1, UP1 Filament of HNRNPA1, NABP2 (SSB1), NABP1(SSB2), or UHRF1.

In one embodiment, a vector includes any of the nucleic acid moleculesdisclosed or described herein.

In one embodiment, a method of modifying a target nucleic acid in acell, includes contacting the cell with a gRNA molecule and an eaCas9fusion molecule, which includes an eaCas9 molecule linked to a templatenucleic acid, such that the gRNA molecule and the eaCas9 fusion moleculeassociate with the target nucleic acid and generate a double strandbreak in the target nucleic acid. In some embodiments, a first overhangand a second overhang in the target nucleic acid are repaired by genecorrection using the template nucleic acid in the eaCas9 fusionmolecule, thereby modifying the target nucleic acid in the cell.

In one embodiment, a method of modifying a target nucleic acid in a cellincludes contacting the cell with a first gRNA molecule, a first eaCas9fusion molecule, which includes a first Cas9 nickase molecule linked toa first template nucleic acid, a second gRNA molecule, and a secondeaCas9 fusion molecule, which includes a second Cas9 nickase moleculelinked to a second template nucleic acid. In some embodiments, the firstgRNA molecule and the first eaCas9 fusion molecule associate with thetarget nucleic acid and generate a first single strand cleavage event ona first strand of the target nucleic acid, and the second gRNA moleculeand the second eaCas9 fusion molecule associate with the target nucleicacid and generate a second single strand cleavage event on a secondstrand of the target nucleic acid, forming a double strand break havinga first overhang and a second overhang. In certain embodiments, thefirst overhang and the second overhang in the target nucleic acid arerepaired by gene correction using the first and second template nucleicacid, thereby modifying the target nucleic acid in the cell.

Headings, including numeric and alphabetical headings and subheadings,are for organization and presentation and are not intended to belimiting.

Other features and advantages of the invention will be apparent from thedetailed description, drawings, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a scheme of generating a Cas9 fusion molecule by covalentconjugation of a single- or multi-cysteine variant Cas9 protein to atemplate nucleic acid that contains a 5′-maleimide-modification.

FIG. 2 depicts the results of an electrophoretic mobility shift assay ofthe products from reacting a 5′-maleimide-modified template nucleic acidwith a cysteine-variant Cas9 protein molecule as described in Example 1.The data indicate the generation of Cas9 fusion molecules containing atleast one covalently attached template nucleic acid.

FIG. 3 depicts a Cas9 fusion molecule formed by covalent conjugation ofa Cas9-HaloTag protein molecule to a template nucleic acid. The singlestranded oligoDNA (ssDNA) is covalently attached to the HaloTagcomponent of the Cas9-HaloTag protein molecule.

FIG. 4A depicts results of the pre-conjugation and pre-annealed methodsfor conjugating a Cas9-HaloTag protein fusion to template sequence usingstandard PAGE analysis for various Cas9 protein fusions provided inTable 9.

FIG. 4B depicts results of the pre-conjugation and pre-annealed methodsfor conjugating a Cas9-HaloTag protein fusion to template sequence usingstandard PAGE analysis for the HGC Cas9 protein fusion provided in Table9.

FIG. 5 depicts the normalized percentages of HDR editing efficiencyusing a nucleofection assay in U2OS cells. The results compare resultsobtained using a Cas9 protein fusion provided in Table 9 conjugated to atemplate nucleic acid with a Cas9-HaloTag protein molecule that is notconjugated to a template nucleic acid. The results demonstrate that theCas9 fusion molecules increase HDR efficiency when conjugated totemplate nucleic acid compared to reactions performed with unconjugatedtemplate nucleic acid.

FIG. 6 depicts a scheme of generating a Cas9 fusion molecule by covalentconjugation of a single- or multi-cysteine variant Cas9 protein moleculeto a template nucleic acid that contains an acrydite-modified templatenucleic acid.

FIG. 7 depicts a scheme generating a Cas9 fusion molecule by covalentconjugation of a N-[ε-Maleimidocaproic acid] hydrazide (EMCH)-modifiedCas9 protein molecule with a 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)-modified template nucleic acid.

FIG. 8 depicts a scheme generating a Cas9 fusion molecule bynon-covalent conjugation of a Cas9 protein molecule, covalently linkedto biotin, and a template nucleic acid, covalently linked to biotin, viathe interaction of the biotin moiety of the Cas9 protein molecule andthe biotin moiety of the template nucleic acid with streptavidin.

DETAILED DESCRIPTION

In order that the invention is understood, certain terms are hereindefined.

Definitions

“Alt-HDR” or “alternative HDR,” or alternative homology-directed repair,as used herein, refers to the process of repairing DNA damage using ahomologous nucleic acid (e.g., an endogenous homologous sequence, e.g.,a sister chromatid, or an exogenous nucleic acid, e.g., a templatenucleic acid). Alt-HDR is distinct from canonical HDR in that theprocess utilizes different pathways from canonical HDR, and can beinhibited by the canonical HDR mediators, RAD51 and BRCA2. Also, alt-HDRuses a single-stranded or nicked homologous nucleic acid for repair ofthe break.

“ALT-NHEJ” or “alternative NHEJ”, or alternative non-homologous endjoining, as used herein, is a type of alternative end joining repairprocess, and utilizes a different pathway than that of canonical NHEJ.In alternative NHEJ, a small degree of resection occurs at the breakends on both sides of the break to reveal single-stranded overhangs.Ligation or annealing of the overhangs results in the deletion ofsequence. ALT-NHEJ is a category that includes microhomology-mediatedend joining (MMEJ), blunt end joining (EJ), and synthesis-dependentmicrohomology-mediated end joining (SD-MMEJ). In MMEJ, microhomologies,or short spans of homologous sequences, e.g., 5 nucleotides or more, onthe single-strand are aligned to guide repair, and leads to the deletionof sequence between the microhomologies.

“Amino acids” as used herein encompasses the canonical amino acids aswell as analogs thereof. “Canonical HDR,” or canonical homology-directedrepair, as used herein, refers to the process of repairing DNA damageusing a homologous nucleic acid (e.g., an endogenous homologoussequence, e.g., a sister chromatid, or an exogenous nucleic acid, e.g.,a template nucleic acid). Canonical HDR typically acts when there hasbeen significant resection at the double-strand break, forming at leastone single stranded portion of DNA. In a normal cell, HDR typicallyinvolves a series of steps such as recognition of the break,stabilization of the break, resection, stabilization of single strandedDNA, formation of a DNA crossover intermediate, resolution of thecrossover intermediate, and ligation. The process requires RAD51 andBRCA2, and the homologous nucleic acid is typically double-stranded.

“Canonical NHEJ”, or canonical non-homologous end joining, as usedherein, refers to the process of repairing double-strand breaks in whichthe break ends are directly ligated. This process does not require ahomologous nucleic acid to guide the repair, and can result in deletionor insertion of one or more nucleotides. This process requires the Kuheterodimer (Ku70/Ku80), the catalytic subunit of DNA-PK (DN-PKcs),and/or DNA ligase XRCC4/LIG4. Unless indicated otherwise, the term “HDR”as used herein encompasses canonical HDR and alt-HDR.

A “Cas9 molecule,” as used herein, refers to a Cas9 polypeptide or anucleic acid encoding a Cas9 polypeptide. A “Cas9 polypeptide” is apolypeptide that can interact with a gRNA molecule and, in concert withthe gRNA molecule, localize to a site comprising a target domain and, incertain embodiments, a PAM sequence. Cas9 molecules include bothnaturally occurring Cas9 molecules and Cas9 molecules and engineered,altered, or modified Cas9 molecules or Cas9 polypeptides that differ,e.g., by at least one amino acid residue, from a reference sequence,e.g., the most similar naturally occurring Cas9 molecule. (The termsaltered, engineered or modified, as used in this context, refer merelyto a difference from a reference or naturally occurring sequence, andimpose no specific process or origin limitations.) A Cas9 molecule maybe a Cas9 polypeptide or a nucleic acid encoding a Cas9 polypeptide. ACas9 molecule may be a nuclease (an enzyme that cleaves both strands ofa double-stranded nucleic acid), a nickase (an enzyme that cleaves onestrand of a double-stranded nucleic acid), or an enzymatically inactive(or dead) Cas9 molecule. A Cas9 molecule having nuclease or nickaseactivity is referred to as an “enzymatically active Cas9 molecule” (an“eaCas9” molecule). A Cas9 molecule lacking the ability to cleave targetnucleic acid is referred to as an “enzymatically inactive Cas9 molecule”(an “eiCas9” molecule). A Cas9 molecule may also be a split Cas9molecule or an inducible Cas9 molecule, as described in more detailbelow.

In certain embodiments, a Cas9 molecule meets one or both of thefollowing criteria: it has at least 20, 30, 40, 50, 55, 60, 65, 70, 75,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 99, or 100% homology with, or it differs by no more than 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35,40, 35, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300,350 or 400, amino acid residues from, the amino acid sequence of areference sequences, e.g., naturally occurring Cas9 molecule.

In certain embodiments, a Cas9 molecule meets one or both of thefollowing criteria: it has at least 20, 30, 40, 50, 55, 60, 65, 70, 75,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 99, or 100% homology with, or it differs by no more than 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35,40, 35, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300,350 or 400, amino acid residues from, the amino acid sequence of areference sequences, e.g., naturally-occurring Cas9 molecule.

In certain embodiments, each domain of the Cas9 molecule (e.g., thedomains named herein) will, independently have: at least 20, 30, 40, 50,55, 60, 65, 70, 75, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,93, 94, 95, 96, 97, 98, 99, or 100% homology with such a domaindescribed herein. In certain embodiments at least 1, 2, 3, 4, 5, of 6domains will have, independently, at least 50, 60, 70, 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%homology with a corresponding domain, while any remaining domains willbe absent, or have less homology to their corresponding naturallyoccurring domains.

In certain embodiments, the Cas9 molecule is a S. pyogenes Cas9 variant.In certain embodiments, the Cas9 variant is the EQR variant. In certainembodiments, the Cas9 variant is the VRER variant. In certainembodiments, the eiCas9 molecule is a S. pyogenes Cas9 variant. Incertain embodiments, the Cas9 variant is the EQR variant. In certainembodiments, the Cas9 variant is the VRER variant. In certainembodiments, a Cas9 system comprises a Cas9 molecule, e.g., a Cas9molecule described herein, e.g., the Cas9 EQR variant or the Cas9 VRERvariant.

In certain embodiments, the Cas9 molecule is a S. aureus Cas9 variant.In certain embodiments, the Cas9 variant is the KKH (E782K/N968K/R1015H)variant (see, e.g., Kleinstiver 2015, the entire contents of which areexpressly incorporated herein by reference). In certain embodiments, theCas9 variant is the E782K/K929R/R1015H variant (see, e.g., Kleinstiver2015). In certain embodiments, the Cas9 variant is theE782K/K929R/N968K/R1015H variant (see, e.g., Kleinstiver 2015). Incertain embodiments the Cas9 variant comprises one or more mutations inone of the following residues: E782, K929, N968, R1015. In certainembodiments the Cas9 variant comprises one or more of the followingmutations: E782K, K929R, N968K, R1015H and R1015Q (see, e.g.,Kleinstiver 2015). In certain embodiments, a Cas9 system comprises aCas9 molecule, e.g., a Cas9 molecule described herein, e.g., the Cas9KKH variant.

A “Cas9 fusion molecule”, “Cas9 fusion protein”, or “Cas9 fusion”, asused herein, refers to a chimeric protein comprising a Cas9 molecule,e.g., Cas9 protein or Cas9 polypeptide, or a fragment thereof, linked toa template nucleic acid. In some embodiments, the template nucleic acidis a nucleic acid, e.g., DNA or RNA. In some embodiments, the templatenucleic acid is single-stranded or double-stranded. In some embodimentsthe template nucleic acid is circular nucleic acid, while in otherembodiments the template nucleic acid is linear nucleic acid. In certainembodiments, the Cas9 fusion molecule comprises a Cas9 moleculecovalently linked to a template nucleic acid. In other embodiments, theCas9 fusion molecule comprises a Cas9 molecule non-covalently linked toa template nucleic acid. In certain embodiments, a Cas9 fusion moleculeis linked to more than one template nucleic acid.

As used herein, the term “Cas9 system” or “gene editing system” refersto a system capable of altering a target nucleic acid by one of many DNArepair pathways. In certain embodiments, the Cas9 system describedherein promotes repair of a target nucleic acid via an HDR pathway. Insome embodiments, a Cas9 system comprises a gRNA and a Cas9 molecule. Inother embodiments, a Cas9 system comprises a gRNA and a Cas9 fusionmolecule. In some embodiments, a Cas9 system further comprises a secondgRNA. In yet another embodiment, a Cas9 system comprises a gRNA, a Cas9molecule, and a second gRNA. In yet other embodiment, a Cas9 systemcomprises a gRNA, a Cas9 fusion molecule, and a second gRNA. In someembodiments, a Cas9 system comprises a gRNA, two Cas9 molecules, and asecond gRNA. In some embodiments, a Cas9 system comprises a gRNA, twoCas9 fusion molecules, and a second gRNA. In some embodiments, a Cas9system comprises a first gRNA, a second gRNA, a first Cas9 molecule, anda second Cas9 molecule. In other embodiments, a Cas9 system comprises afirst gRNA, a second gRNA, a first Cas9 fusion molecule, and a secondCas9 fusion molecule. In some embodiments, a Cas9 system furthercomprises a template nucleic acid. In other embodiments, a Cas9 systemfurther comprises a template nucleic acid provided by the Cas9 fusionmolecule.

As used herein, the term “cleavage event” refers to a break in a nucleicacid molecule. A cleavage event may be a single-strand cleavage event,or a double-strand cleavage event. A single-strand cleavage event mayresult in a 5′ overhang or a 3′ overhang. A double-stranded cleavageevent may result in blunt ends, two 5′ overhangs, or two 3′ overhangs.

A disorder “caused by” a mutation, as used herein, refers to a disorderthat is made more likely or severe by the presence of the mutation,compared to a subject that does not have the mutation. The mutation neednot be the only cause of a disorder, i.e., the disorder can still becaused by the mutation even if other causes, such as environmentalfactors or lifestyle factors, contribute causally to the disorder. Inembodiments, the disorder is caused by the mutation if the mutation is amedically recognized risk factor for developing the disorder, and/or ifa study has found that the mutation contributes causally to developmentof the disorder.

The term “covalent”, as used herein, refers to a form of chemicalbonding characterized by the sharing of one or more pairs of electronsbetween two components, producing a mutual attraction that holds the twocomponents together. The sharing of the one or more pairs of electronsbetween two components may either be direct (e.g., via reactive groupson the surface the two components, e.g., a Cas9 polypeptide and atemplate nucleic acid) or indirect (via a linker molecule).

“Derived from”, as used herein, refers to the source or origin of amolecular entity, e.g., a nucleic acid or protein. The source of amolecular entity may be naturally-occurring, recombinant, unpurified, ora purified molecular entity. For example, a polypeptide that is derivedfrom a second polypeptide comprises an amino acid sequence that isidentical or substantially similar, e.g., is more than 50% homologousto, the amino acid sequence of the second protein. The derived molecularentity, e.g., a nucleic acid or protein, can comprise one or moremodifications, e.g., one or more amino acid or nucleotide changes.

“Domain,” as used herein, is used to describe a segment of, or a portionof a protein or nucleic acid. Unless otherwise indicated, a domain isnot required to have any specific functional property.

Calculations of homology or sequence identity between two sequences (theterms are used interchangeably herein) are performed as follows. Thesequences are aligned for optimal comparison purposes (e.g., gaps can beintroduced in one or both of a first and a second amino acid or nucleicacid sequence for optimal alignment and non-homologous sequences can bedisregarded for comparison purposes). The optimal alignment isdetermined as the best score using the GAP program in the GCG softwarepackage with a Blossum 62 scoring matrix with a gap penalty of 12, a gapextend penalty of 4, and a frame shift gap penalty of 5. The amino acidresidues or nucleotides at corresponding amino acid positions ornucleotide positions are then compared. When a position in the firstsequence is occupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position. The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences.

As used herein, the term “endogenous” gene, “endogenous” nucleic acid,or “endogenous” homologous region refers to a native gene, nucleic acid,or region of a gene, which is in its natural location in the genome,e.g., chromosome or plasmid, of a cell. In contrast, the term“exogenous” gene or “exogenous” nucleic acid refers to a gene, nucleicacid, or region of a gene which is not native within a cell, but whichis introduced into the cell during the methods of the invention. Anexogenous gene or exogenous nucleic acid may be homologous to, oridentical to, an endogenous gene or an endogenous nucleic acid.

As used herein, the term “endogenous homologous region” refers to anendogenous template nucleic acid sequence which is homologous to atleast a portion of a target gene, and which can be used in conjunctionwith a Cas9 molecule and a gRNA molecule to modify, e.g., correct, asequence of the target gene. In one embodiment, the endogenoushomologous region is DNA. In another embodiment, the endogenoushomologous region is double stranded DNA. In another embodiment, theendogenous homologous region is single stranded DNA. In one embodiment,the endogenous homologous region is at least 70%, 75%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 875, 885, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,9%, 98%, or 99% homologous to at least a portion of the target gene.

As used herein, the term “enzymatically inactive Cas9” (“eiCas9”) oreiCas9 polypeptide refers to Cas9 molecules having no, or nosubstantial, cleavage activity. For example, an eiCas9 molecule oreiCas9 polypeptide can lack cleavage activity or have substantiallyless, e.g., less than 20, 10, 5, 1 or 0.1% of the cleavage activity of areference Cas9 molecule or eiCas9 polypeptide, as measured by an assaydescribed herein.

In one embodiment, a Cas9 molecule is an eiCas9 molecule comprising oneor more differences in a RuvC domain and/or in an HNH domain as comparedto a reference Cas9 molecule, and the eiCas9 molecule does not cleave anucleic acid, or cleaves with significantly less efficiency than doeswild type, e.g., when compared with wild type in a cleavage assay, e.g.,as described herein, cuts with less than 50, 25, 10, or 1% of areference Cas9 molecule, as measured by an assay described herein. Thereference Cas9 molecule can be a naturally occurring unmodified Cas9molecule, e.g., a naturally occurring Cas9 molecule such as a Cas9molecule of S. pyogenes, S. thermophilus, S. aureus, C. jejuni or N.meningitidis. In one embodiment, the reference Cas9 molecule is thenaturally occurring Cas9 molecule having the closest sequence identityor homology. In one embodiment, the eiCas9 molecule lacks substantialcleavage activity associated with a RuvC domain and cleavage activityassociated with an HNH domain.

Whether or not a particular sequence, e.g., a substitution, may affectone or more activity, such as targeting activity, cleavage activity,etc., can be evaluated or predicted, e.g., by evaluating whether themutation is conservative. In one embodiment, a “non-essential” aminoacid residue, as used in the context of a Cas9 molecule, is a residuethat can be altered from the wild-type sequence of a Cas9 molecule,e.g., a naturally occurring Cas9 molecule, e.g., an eaCas9 molecule,without abolishing or more preferably, without substantially altering aCas9 activity (e.g., cleavage activity), whereas changing an “essential”amino acid residue results in a substantial loss of activity (e.g.,cleavage activity).

Although an enzymatically inactive (eiCas9) Cas9 molecule itself canblock transcription when recruited to early regions in the codingsequence, more robust repression can be achieved by fusing atranscriptional repression domain (for example KRAB, SID or ERD) to theCas9 and recruiting it to the target knockdown position, e.g., within1000 bp of sequence 3′ of the start codon or within 500 bp of a promoterregion 5′ of the start codon of a gene. It is likely that targetingDNAseI hypersensitive sites (DHSs) of the promoter may yield moreefficient gene repression or activation because these regions are morelikely to be accessible to the Cas9 protein and are also more likely toharbor sites for endogenous transcription factors. Especially for generepression, it is contemplated herein that blocking the binding site ofan endogenous transcription factor would aid in downregulating geneexpression. In one embodiment, one or more eiCas9 molecules may be usedto block binding of one or more endogenous transcription factors. Inanother embodiment, an eiCas9 molecule can be fused to a chromatinmodifying protein. Altering chromatin status can result in decreasedexpression of the target gene. One or more eiCas9 molecules fused to oneor more chromatin modifying proteins may be used to alter chromatinstatus.

As used herein, “error-prone” repair refers to a DNA repair process thathas a higher tendency to introduce mutations into the site beingrepaired. For instance, alt-NHEJ and SSA are error-prone pathways;C-NHEJ is also error prone because it sometimes leads to the creation ofa small degree of alteration of the site (even though in some instancesC-NHEJ results in error-free repair); and HR, alt-HR, and SSA in thecase of a single-strand oligo donor are not error-prone. As used herein,the term “gRNA molecule” or “gRNA” refers to a guide RNA which iscapable of targeting a Cas9 molecule to a target nucleic acid. In oneembodiment, the term “gRNA molecule” refers to a guide ribonucleic acid.In another embodiment, the term “gRNA molecule” refers to a nucleic acidencoding a gRNA. In one embodiment, a gRNA molecule is non-naturallyoccurring. In one embodiment, a gRNA molecule is a synthetic gRNAmolecule.

“Governing gRNA molecule,” as used herein, refers to a gRNA moleculethat comprises a targeting domain that is complementary to a targetdomain on a nucleic acid that comprises a sequence that encodes acomponent of the CRISPR/Cas system that is introduced into a cell orsubject. A governing gRNA does not target an endogenous cell or subjectsequence. In one embodiment, a governing gRNA molecule comprises atargeting domain that is complementary with a target sequence on: (a) anucleic acid that encodes a Cas9 molecule; (b) a nucleic acid thatencodes a gRNA molecule which comprises a targeting domain that targetsa target gene (a target gene gRNA); or on more than one nucleic acidthat encodes a CRISPR/Cas component, e.g., both (a) and (b). In oneembodiment, a nucleic acid molecule that encodes a CRISPR/Cas component,e.g., that encodes a Cas9 molecule or a target gene gRNA molecule,comprises more than one target domain that is complementary with agoverning gRNA targeting domain. While not wishing to be bound bytheory, it is believed that a governing gRNA molecule complexes with aCas9 molecule and results in Cas9 mediated inactivation of the targetednucleic acid, e.g., by cleavage or by binding to the nucleic acid, andresults in cessation or reduction of the production of a CRISPR/Cassystem component. In one embodiment, the Cas9 molecule forms twocomplexes: a complex comprising a Cas9 molecule with a target gene gRNAmolecule, which complex will alter the target gene; and a complexcomprising a Cas9 molecule with a governing gRNA molecule, which complexwill act to prevent further production of a CRISPR/Cas system component,e.g., a Cas9 molecule or a target gene gRNA molecule. In one embodiment,a governing gRNA molecule/Cas9 molecule complex binds to or promotescleavage of a control region sequence, e.g., a promoter, operably linkedto a sequence that encodes a Cas9 molecule, a sequence that encodes atranscribed region, an exon, or an intron, for the Cas9 molecule. In oneembodiment, a governing gRNA molecule/Cas9 molecule complex binds to orpromotes cleavage of a control region sequence, e.g., a promoter,operably linked to a gRNA molecule, or a sequence that encodes the gRNAmolecule. In one embodiment, the governing gRNA molecule, e.g., aCas9-targeting governing gRNA molecule, or a target gene gRNA-targetinggoverning gRNA molecule, limits the effect of the Cas9 molecule/targetgene gRNA molecule complex-mediated gene targeting. In one embodiment, agoverning gRNA places temporal, level of expression, or other limits, onactivity of the Cas9 molecule/target gene gRNA molecule complex. In oneembodiment, a governing gRNA reduces off-target or other unwantedactivity. In one embodiment, a governing gRNA molecule inhibits, e.g.,entirely or substantially entirely inhibits, the production of acomponent of the Cas9 system and thereby limits, or governs, itsactivity.

“HDR”, or homology-directed repair, as used herein, refers to theprocess of repairing DNA damage using a homologous nucleic acid (e.g.,an endogenous nucleic acid, e.g., a sister chromatid, or an exogenousnucleic acid, e.g., a template nucleic acid). HDR typically occurs whenthere has been significant resection at a double-strand break, formingat least one single stranded portion of DNA. HDR is a category thatincludes, for example, single-strand annealing (SSA), homologousrecombination (HR), single strand template repair (SST-R), and a third,not yet fully characterized alternative homologous recombination(alt-HR) DNA repair pathway. In some embodiments, HDR includes geneconversion and gene correction. In some embodiments, the term HDR doesnot encompass canonical NHEJ (C-NHEJ). In some embodiments, the term HDRdoes not encompass alternative non-homologous end joining (Alt-NHEJ)(e.g., blunt end-joining (blunt EJ), (micro homology mediated endjoining (MMEJ), and synthesis dependent microhomology-mediated endjoining (SD-MMEJ)).

The terms “homology” or “identity,” as used interchangeably herein,refer to sequence identity between two amino acid sequences or twonucleic acid sequences, with identity being a more strict comparison.The phrases “percent identity or homology” and “% identity or homology”refer to the percentage of sequence identity found in a comparison oftwo or more amino acid sequences or nucleic acid sequences. Two or moresequences can be anywhere from 0-100% identical, or any value therebetween. Identity can be determined by comparing a position in eachsequence that can be aligned for purposes of comparison to a referencesequence. When a position in the compared sequence is occupied by thesame nucleotide base or amino acid, then the molecules are identical atthat position. A degree of identity of amino acid sequences is afunction of the number of identical amino acids at positions shared bythe amino acid sequences. A degree of identity between nucleic acidsequences is a function of the number of identical or matchingnucleotides at positions shared by the nucleic acid sequences. A degreeof homology of amino acid sequences is a function of the number of aminoacids at positions shared by the polypeptide sequences.

Calculations of homology or sequence identity between two sequences (theterms are used interchangeably herein) are performed as follows. Thesequences are aligned for optimal comparison purposes (e.g., gaps can beintroduced in one or both of a first and a second amino acid or nucleicacid sequence for optimal alignment and non-homologous sequences can bedisregarded for comparison purposes). The optimal alignment isdetermined as the best score using the GAP program in the GCG softwarepackage with a Blossum 62 scoring matrix with a gap penalty of 12, a gapextend penalty of 4, and a frame shift gap penalty of 5. The amino acidresidues or nucleotides at corresponding amino acid positions ornucleotide positions are then compared. When a position in the firstsequence is occupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position. The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences.

“Gene conversion”, as used herein, refers to the process of repairingDNA damage by homology directed recombination (HDR) using an endogenousnucleic acid, e.g., a sister chromatid or a plasmid, as a templatenucleic acid. Without being bound by theory, in some embodiments, BRCA1,BRCA2 and/or RAD51 are believed to be involved in gene conversion. Insome embodiments, the endogenous nucleic acid is a nucleic acid sequencehaving homology, e.g., significant homology, with a fragment of DNAproximal to the site of the DNA lesion or mutation. In some embodiments,the template is not an exogenous nucleic acid.

“Gene correction”, as used herein, refers to the process of repairingDNA damage by homology directed recombination using an exogenous nucleicacid, e.g., a donor template nucleic acid. In some embodiments, theexogenous nucleic acid is single-stranded. In some embodiments, theexogenous nucleic acid is double-stranded. In one embodiment, the donortemplate nucleic acid is a circular nucleic acid sequence. In anotherembodiment, the donor template nucleic acid is a linear nucleic acidsequence.

“Homologous recombination” or “HR” refers to a type of HDR DNA-repairwhich typically acts occurs when there has been significant resection atthe double-strand break, forming at least one single stranded portion ofDNA. In a normal cell, HR typically involves a series of steps such asrecognition of the break, stabilization of the break, resection,stabilization of single stranded DNA, formation of a DNA crossoverintermediate, resolution of the crossover intermediate, and ligation.The process requires RAD51 and BRCA2, and the homologous nucleic acid istypically double-stranded. In some embodiments, homologous recombinationincludes gene conversion.

“Ligand acceptor molecule,” as used herein, refers to a substance ormolecule that specifically interacts non-covalently with at least oneligand. In one embodiment, the ligand acceptor molecule is streptavidin.

The term “ligand,” as used herein, refers to a substance or moleculethat specifically interacts with another substance or molecule (e.g., aligand acceptor molecule). In one embodiment, the ligand is biotin. Insome embodiments, the ligand is a high-affinity ligand (e.g., a ligandthat has high affinity for its receptor.

The term “linked” or “linkage” as used herein means an interactionbetween molecules or parts of molecules. Two molecules that are linkedmay be covalently linked or non-covalently linked.

The term “linker”, as used herein, refers to a molecule whichfacilitates an interaction between molecules or parts of molecules. Inone embodiment, a linker is a polypeptide linker. In another embodiment,a linker is a nucleic acid linker.

The term “peptide linker” or “polypeptide linker” as used herein means apeptide or polypeptide comprising two or more amino acids residuesjoined by peptide bonds. Such peptide or polypeptide linkers are wellknown in the art. Linkers comprise naturally occurring and/ornon-naturally occurring peptides or polypeptides.

“Modulator,” as used herein, refers to an entity, e.g., a compound, thatcan alter the activity (e.g., enzymatic activity, transcriptionalactivity, or translational activity), amount, distribution, or structureof a subject molecule or genetic sequence. In one embodiment, modulationcomprises cleavage, e.g., breaking of a covalent or non-covalent bond,or the forming of a covalent or non-covalent bond, e.g., the attachmentof a moiety, to the subject molecule. In one embodiment, a modulatoralters the, three dimensional, secondary, tertiary, or quaternarystructure, of a subject molecule. A modulator can increase, decrease,initiate, or eliminate a subject activity.

As used herein, the term “mutation” refers to a change in the sequenceof a nucleic acid as compared to a wild-type sequence of the nucleicacid, resulting a variant form of the nucleic acid. A mutation in anucleic acid may be caused by the alteration of a single base pair inthe nucleic acid, or the insertion, deletion, or rearrangement of largersections of the nucleic acid. A mutation in a gene may result invariants of the protein encoded by the gene which are associated withgenetic disorders.

The term “non-covalent bond” refers to a variety of interactions betweenmolecules or parts of molecules that are not covalent in nature, whichprovide force to hold the molecules or parts of molecules togetherusually in a specific orientation or conformation. Such non-covalentinteractions include inter alia ionic bonds, hydrophobic interactions,hydrogen bonds, Van-der-Waals forces, and dipole-dipole bonds.

“Non-homologous end joining” or “NHEJ,” as used herein, refers toligation mediated repair and/or non-template mediated repair includingcanonical NHEJ (cNHEJ), alternative NHEJ (altNHEJ),microhomology-mediated end joining (MMEJ), single-strand annealing(SSA), and synthesis-dependent microhomology-mediated end joining(SD-MMEJ). Unless indicate otherwise, “NHEJ” as used herein encompassescanonical NHEJ, alt-NHEJ, MMEJ, SSA and SD-MMEJ.

“Polypeptide,” as used herein, refers to a polymer of amino acids.

The term “protein”, as used herein, is intended to refer to abiomolecule comprised of amino acids arranged in the form of apolypeptide. A protein may be a full-length protein, or a fragmentthereof.

As used herein, the term “processing,” with respect to overhangs, refersto either the endonucleolytic processing or the exonucleolyticprocessing of a break in a nucleic acid molecule. In one embodiment,processing of a 5′ overhang in a nucleic acid molecule may result in a3′ overhang. In another embodiment, processing of a 3′ overhang in anucleic acid molecule may result in a 5′ overhang.

A “reference molecule,” as used herein, refers to a molecule to which amodified or candidate molecule is compared. For example, a referenceCas9 molecule refers to a Cas9 molecule to which a modified or candidateCas9 molecule is compared. The modified or candidate molecule may mecompared to the reference molecule on the basis of sequence (e.g., themodified or candidate may have X % sequence identity or homology withthe reference molecule) or activity (e.g., the modified or candidatemolecule may have X % of the activity of the reference molecule). Forexample, where the reference molecule is a Cas9 molecule, a modified orcandidate may be characterized as having no more than 10% of thenuclease activity of the reference Cas9 molecule. Examples of referenceCas9 molecules include naturally occurring unmodified Cas9 molecules,e.g., a naturally occurring Cas9 molecule from S. pyogenes, S. aureus,S. thermophilus or N. meningitidis. In certain embodiments, thereference Cas9 molecule is the naturally occurring Cas9 molecule havingthe closest sequence identity or homology with the modified or candidateCas9 molecule to which it is being compared. In certain embodiments, thereference Cas9 molecule is a parental molecule having a naturallyoccurring or known sequence on which a mutation has been made to arriveat the modified or candidate Cas9 molecule.

“Replacement,” or “replaced,” as used herein with reference to amodification of a molecule does not require a process limitation butmerely indicates that the replacement entity is present.

“Resection”, as used herein, refers to exonuclease-mediated digestion ofone strand of a double-stranded DNA molecule, which results in asingle-stranded overhang. Resection may occur, e.g., on one or bothsides of a double-stranded break. Resection can be measured by, forinstance, extracting genomic DNA, digesting it with an enzyme thatselectively degrades dsDNA, and performing quantitative PCR usingprimers spanning the DSB site, e.g., as described herein.

“SSA” or “Single-strand Annealing”, as used herein, refers to theprocess where RAD52 as opposed to RAD51 in the HR pathways, binds to thesingle stranded portion of DNA and promotes annealing of the two singlestranded DNA segments at repetitive regions. Once RAD52 binds XFP/ERCC1removes DNA flaps to make the DNA more suitable for ligation.

“Subject,” as used herein, may mean either a human or non-human animal.The term includes, but is not limited to, mammals (e.g., humans, otherprimates, pigs, rodents (e.g., mice and rats or hamsters), rabbits,guinea pigs, cows, horses, cats, dogs, sheep, and goats). In oneembodiment, the subject is a human. In another embodiment, the subjectis poultry. In another embodiment, the subject is piscine. In certainembodiments, the subject is a human, and in certain of these embodimentsthe human is an infant, child, young adult, or adult.

As used herein, the terms “target nucleic acid” or “target gene” referto a nucleic acid which is being targeted for alteration, e.g., by genecorrection, by a Cas9 system described herein. In certain embodiments, atarget nucleic acid comprises one gene. In certain embodiments, a targetnucleic acid may comprise one or more genes, e.g., two genes, threegenes, four genes, or five genes. In one embodiment, a target nucleicacid may comprise a promoter region, or control region, of a gene. Inone embodiment, a target nucleic acid may comprise an intron of a gene.In another embodiment, a target nucleic acid may comprise an exon of agene. In one embodiment, a target nucleic acid may comprise a codingregion of gene. In one embodiment, a target nucleic acid may comprise anon-coding region of a gene.

“Target position” as used herein, refers to a site on a target nucleicacid that is modified by a Cas9 molecule-dependent or a Cas9 fusionmolecule-dependent process. For example, the target position can bemodified by a Cas9 molecule-mediated cleavage (or a Cas9 fusionmolecule-mediated cleavage) of the target nucleic acid and templatenucleic acid directed modification, e.g., correction, of the targetposition. In one embodiment, a target position can be a site between twonucleotides, e.g., adjacent nucleotides, on the target nucleic acid intowhich one or more nucleotides is added based on homology with a templatenucleic acid. The target position may comprise one or more nucleotidesthat are altered, e.g., corrected, based on homology with a templatenucleic acid. In another embodiment, the target position may compriseone or more nucleotides that are deleted based on homology with atemplate nucleic acid. In one embodiment, the target position is withina “target sequence” (e.g., the sequence to which the gRNA binds). In oneembodiment, a target position is upstream or downstream of a targetsequence (e.g., the sequence to which the gRNA binds).

“Target region,” “target domain,” or “target sequence,” as used herein,is a nucleic acid sequence that comprises a target position and at leastone nucleotide position outside the target position. In certainembodiments, the target position is flanked by sequences of the targetposition region, i.e., the target position is disposed in the targetposition region such that there are target position region sequencesboth 5′ and 3′ to the target position. In certain embodiments, thetarget position region provides sufficient sequences on each side (i.e.,5′ and 3′) of the target position to allow gene correction of the targetposition, wherein the gene correction uses an exogenous sequencehomologous with the target position region as a template.

A “template nucleic acid,” “exogenous homologous region,” “donor nucleicacid,” “exogenous template,” or “donor template” as that term is usedherein, refers to a nucleic acid sequence which can be used inconjunction with a Cas9 molecule (or a Cas9 fusion molecule) and a gRNAmolecule and services as a guide for altering the structure of a targetposition. In some embodiments, the template nucleic acid is homologousto at least a portion of a target gene, and which can be used inconjunction with a Cas9 molecule and a gRNA molecule to modify, e.g.,correct, a sequence of the target gene. In one embodiment, the targetnucleic acid is modified to have the some or all of the sequence of thetemplate nucleic acid, typically at or near cleavage site(s). In someembodiments, the template nucleic acid is a nucleic acid, e.g., DNA orRNA. In one embodiment, the template nucleic acid is single stranded. Inan alternate embodiment, the template nucleic acid is double stranded.In one embodiment, the template nucleic acid is DNA, e.g., doublestranded DNA. In an alternate embodiment, the template nucleic acid issingle stranded DNA. In one embodiment, the template nucleic acid isencoded on the same vector backbone, e.g., AAV genome, plasmid DNA, asthe Cas9 and gRNA. In one embodiment, the template nucleic acid isexcised from a vector backbone in vivo, e.g., it is flanked by gRNArecognition sequences. In one embodiment, the template nucleic acidcomprises endogenous genomic sequence. In one embodiment, the templatenucleic acid is an RNA. In some embodiments the template nucleic acid iscircular nucleic acid. In other embodiments, the template nucleic acidis linear nucleic acid.

In one embodiment, the template nucleic acid alters the structure of thetarget position by participating in a homology directed repair event,e.g., a gene correction event. In one embodiment, the template nucleicacid alters the sequence of the target position. In one embodiment, thetemplate nucleic acid results in the incorporation of a modified, ornon-naturally occurring base into the target nucleic acid.

Typically, the template sequence undergoes a breakage mediated orcatalyzed recombination with the target sequence. In one embodiment, thetemplate nucleic acid includes sequence that corresponds to a site onthe target sequence that is cleaved by an eaCas9 mediated cleavageevent. In one embodiment, the template nucleic acid includes sequencethat corresponds to both, a first site on the target sequence that iscleaved in a first Cas9 mediated event, and a second site on the targetsequence that is cleaved in a second Cas9 mediated event.

In one embodiment, the template nucleic acid can include sequence whichresults in an alteration in the coding sequence of a translatedsequence, e.g., one which results in the substitution of one amino acidfor another in a protein product, e.g., transforming a mutant alleleinto a wild type allele, transforming a wild type allele into a mutantallele, and/or introducing a stop codon, insertion of an amino acidresidue, deletion of an amino acid residue, or a nonsense mutation.

In other embodiments, the template nucleic acid can include sequencewhich results in an alteration in a non-coding sequence, e.g., analteration in an exon or in a 5′ or 3′ non-translated or non-transcribedregion. Such alterations include an alteration in a control element,e.g., a promoter, enhancer, and an alteration in a cis-acting ortrans-acting control element.

A template nucleic acid having homology with a target position in agene, e.g., a gene described herein, can be used to alter the structureof a target sequence. The template sequence can be used to alter anunwanted structure, e.g., an unwanted or mutant nucleotide.

A template nucleic acid typically comprises the following components:

[5′ homology arm]-[replacement sequence]-[3′ homology arm].

The homology arms provide for recombination into the chromosome, thusreplacing the undesired element, e.g., a mutation or signature, with areplacement sequence, e.g., the desired, or corrected sequence. In oneembodiment, the homology arms flank the most distal cleavage sites.

In one embodiment, the 3′ end of the 5′ homology arm is the positionnext to the 5′ end of the replacement sequence. In one embodiment, the5′ homology arm can extend at least 10, 20, 30, 40, 50, 100, 200, 300,400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 3000, 4000, or 5000nucleotides 5′ from the 5′ end of the replacement sequence.

In one embodiment, the 5′ end of the 3′ homology arm is the positionnext to the 3′ end of the replacement sequence. In one embodiment, the3′ homology arm can extend at least 10, 20, 30, 40, 50, 100, 200, 300,400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 3000, 4000, or 5000nucleotides 3′ from the 3′ end of the replacement sequence.

In one embodiment, to correct a mutation, the homology arms, e.g., the5′ and 3′ homology arms, may each comprise about 1000 base pairs (bp) ofsequence flanking the most distal gRNAs (e.g., 1000 bp of sequence oneither side of the mutation).

It is contemplated herein that one or both homology arms may beshortened to avoid including certain sequence repeat elements, e.g., Alurepeats or LINE elements. For example, a 5′ homology arm may beshortened to avoid a sequence repeat element. In other embodiments, a 3′homology arm may be shortened to avoid a sequence repeat element. Insome embodiments, both the 5′ and the 3′ homology arms may be shortenedto avoid including certain sequence repeat elements.

It is contemplated herein that template nucleic acids for correcting amutation may be designed for use as a single-stranded oligonucleotide,e.g., a single-stranded oligodeoxynucleotide (ssODN). When using assODN, 5′ and 3′ homology arms may range up to about 200 base pairs (bp)in length, e.g., at least 25, 50, 75, 100, 125, 150, 175, or 200 bp inlength. Longer homology arms are also contemplated for ssODNs asimprovements in oligonucleotide synthesis continue to be made. In someembodiments, a longer homology arm is made by a method other thanchemical synthesis, e.g., by denaturing a long double stranded nucleicacid and purifying one of the strands, e.g., by affinity for astrand-specific sequence anchored to a solid substrate.

While not wishing to be bound by theory, in some embodiments HDRproceeds more efficiently when the template nucleic acid has extendedhomology 5′ to a nick, (i.e., in the 5′ direction of the nicked strand).A nick, as referred to herein, refers to a single strand break in anucleic acid. Accordingly, in some embodiments, the template nucleicacid has a longer homology arm and a shorter homology arm, wherein thelonger homology arm can anneal 5′ of the nick. In some embodiments, thearm that can anneal 5′ to the nick is at least 25, 50, 75, 100, 125,150, 175, or 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000,3000, 4000, or 5000 nucleotides from the nick or the 5′ or 3′ end of thereplacement sequence. In some embodiments, the arm that can anneal 5′ tothe nick is at least 10%, 20%, 30%, 40%, or 50% longer than the arm thatcan anneal 3′ to the nick. In some embodiments, the arm that can anneal5′ to the nick is at least 2×, 3×, 4×, or 5× longer than the arm thatcan anneal 3′ to the nick. Depending on whether a ssDNA template cananneal to the intact strand or the nicked strand, the homology arm thatanneals 5′ to the nick may be at the 5′ end of the ssDNA template or the3′ end of the ssDNA template, respectively.

Similarly, in some embodiments, the template nucleic acid has a 5′homology arm, a replacement sequence, and a 3′ homology arm, such thatthe template nucleic acid has extended homology to the 5′ of the nick.For example, the 5′ homology arm and 3′ homology arm may besubstantially the same length, but the replacement sequence may extendfarther 5′ of the nick than 3′ of the nick. In some embodiments, thereplacement sequence extends at least 10%, 20%, 30%, 40%, 50%, 2×, 3×,4×, or 5× further to the 5′ end of the nick than the 3′ end of the nick.

While not wishing to be bound by theory, in some embodiments HDRproceeds more efficiently when the template nucleic acid is centered onthe nick. Accordingly, in some embodiments, the template nucleic acidhas two homology arms that are essentially the same size. For instance,the first homology arm of a template nucleic acid may have a length thatis within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the secondhomology arm of the template nucleic acid.

Similarly, in some embodiments, the template nucleic acid has a 5′homology arm, a replacement sequence, and a 3′ homology arm, such thatthe template nucleic acid extends substantially the same distance oneither side of the nick. For example, the homology arms may havedifferent lengths, but the replacement sequence may be selected tocompensate for this. For example, the replacement sequence may extendfurther 5′ from the nick than it does 3′ of the nick, but the homologyarm 5′ of the nick is shorter than the homology arm 3′ of the nick, tocompensate. The converse is also possible, e.g., that the replacementsequence may extend further 3′ from the nick than it does 5′ of thenick, but the homology arm 3′ of the nick is shorter than the homologyarm 5′ of the nick, to compensate.

A “variant Cas9 molecule,” as used herein refers to a Cas9 molecule withat least one modification, e.g., a mutation or chemical modification toat least one amino acid residue of the wild-type Cas9 molecule.

Exemplary Arrangements of Linear Nucleic Acid Template Systems

In one embodiment, the template nucleic acid is double stranded. In oneembodiment, the template nucleic acid is single stranded. In oneembodiment, the nucleic acid template system comprises a single strandedportion and a double stranded portion. In one embodiment, the templatenucleic acid comprises about 50 to 100, e.g., 55 to 95, 60 to 90, 65 to85, or 70 to 80, base pairs, homology on either side of the nick and/orreplacement sequence. In one embodiment, the template nucleic acidcomprises about 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 basepairs homology 5′ of the nick or replacement sequence, 3′ of the nick orreplacement sequence, or both 5′ and 3′ of the nick or replacementsequences.

In one embodiment, the template nucleic acid comprises about 150 to 200,e.g., 155 to 195, 160 to 190, 165 to 185, or 170 to 180, base pairshomology 3′ of the nick and/or replacement sequence. In one embodiment,the template nucleic acid comprises about 150, 155, 160, 165, 170, 175,180, 185, 190, 195, or 200 base pairs homology 3′ of the nick orreplacement sequence. In one embodiment, the template nucleic acidcomprises less than about 100, 90, 80, 70, 60, 50, 40, 30, 20, 15, or 10base pairs homology 5′ of the nick or replacement sequence.

In one embodiment, the template nucleic acid comprises about 150 to 200,e.g., 155 to 195, 160 to 190, 165 to 185, or 170 to 180, base pairshomology 5′ of the nick and/or replacement sequence. In one embodiment,the template nucleic acid comprises about 150, 155, 160, 165, 170, 175,180, 185, 190, 195, or 200 base pairs homology 5′ of the nick orreplacement sequence. In one embodiment, the template nucleic acidcomprises less than about 100, 90, 80, 70, 60, 50, 40, 30, 20, 15, or 10base pairs homology 3′ of the nick or replacement sequence.

Exemplary Template Nucleic Acids

In one embodiment, the template nucleic acid is a single strandednucleic acid. In another embodiment, the template nucleic acid is adouble stranded nucleic acid. In some embodiments, the template nucleicacid comprises a nucleotide sequence, e.g., of one or more nucleotides,that will be added to or will create a change in, or correct thesequence of the target nucleic acid to a desired sequence. In otherembodiments, the template nucleic acid comprises a nucleotide sequencethat may be used to modify the target position. In other embodiments,the template nucleic acid comprises a nucleotide sequence, e.g., of oneor more nucleotides, that corresponds to wild type sequence of thetarget nucleic acid, e.g., of the target position.

The template nucleic acid may comprise a replacement sequence. Areplacement sequence, as the term is used herein, refers to a sequencewhich will serve as the template for making the desired change, orcorrection, in the target nucleic acid. The replacement sequence ishomologous, but not identical to, the target nucleic acid. In someembodiments, the template nucleic acid comprises a 5′ homology arm. Inother embodiments, the template nucleic acid comprises a 3′ homologyarm.

In embodiments, the template nucleic acid is linear double stranded DNA.The length may be, e.g., about 150-200 base pairs, e.g., about 150, 160,170, 180, 190, or 200 base pairs. The length may be, e.g., at least 150,160, 170, 180, 190, or 200 base pairs. In some embodiments, the lengthis no greater than 150, 160, 170, 180, 190, or 200 base pairs. In someembodiments, a double stranded template nucleic acid has a length ofabout 160 base pairs, e.g., about 155-165, 150-170, 140-180, 130-190,120-200, 110-210, 100-220, 90-230, or 80-240 base pairs.

The template nucleic acid can be linear single stranded DNA. Inembodiments, the template nucleic acid is (i) linear single stranded DNAthat can anneal to the nicked strand of the target nucleic acid, (ii)linear single stranded DNA that can anneal to the intact strand of thetarget nucleic acid, (iii) linear single stranded DNA that can anneal tothe transcribed strand of the target nucleic acid, (iv) linear singlestranded DNA that can anneal to the non-transcribed strand of the targetnucleic acid, or more than one of the preceding. The length may be,e.g., about 150-200 nucleotides, e.g., about 150, 160, 170, 180, 190, or200 nucleotides. The length may be, e.g., at least 150, 160, 170, 180,190, or 200 nucleotides. In some embodiments, the length is no greaterthan 150, 160, 170, 180, 190, or 200 nucleotides. In some embodiments, asingle stranded template nucleic acid has a length of about 160nucleotides, e.g., about 155-165, 150-170, 140-180, 130-190, 120-200,110-210, 100-220, 90-230, or 80-240 nucleotides.

In some embodiments, the template nucleic acid is circular doublestranded DNA, e.g., a plasmid. In some embodiments, the template nucleicacid comprises about 500 to 1000 base pairs of homology on either sideof the replacement sequence and/or the nick. In some embodiments, thetemplate nucleic acid comprises about 300, 400, 500, 600, 700, 800, 900,1000, 1500, or 2000 base pairs of homology 5′ of the nick or replacementsequence, 3′ of the nick or replacement sequence, or both 5′ and 3′ ofthe nick or replacement sequence. In some embodiments, the templatenucleic acid comprises at least 300, 400, 500, 600, 700, 800, 900, 1000,1500, or 2000 base pairs of homology 5′ of the nick or replacementsequence, 3′ of the nick or replacement sequence, or both 5′ and 3′ ofthe nick or replacement sequence. In some embodiments, the templatenucleic acid comprises no more than 300, 400, 500, 600, 700, 800, 900,1000, 1500, or 2000 base pairs of homology 5′ of the nick or replacementsequence, 3′ of the nick or replacement sequence, or both 5′ and 3′ ofthe nick or replacement sequence.

In some embodiments, the template nucleic acid is an adenovirus vector,e.g., an AAV vector, e.g., a ssDNA molecule of a length and sequencethat allows it to be packaged in an AAV capsid. The vector may be, e.g.,less than 5 kb and may contain an ITR sequence that promotes packaginginto the capsid. The vector may be integration-deficient. In someembodiments, the template nucleic acid comprises about 150 to 1000nucleotides of homology on either side of the replacement sequenceand/or the nick. In some embodiments, the template nucleic acidcomprises about 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000,1500, or 2000 nucleotides 5′ of the nick or replacement sequence, 3′ ofthe nick or replacement sequence, or both 5′ and 3′ of the nick orreplacement sequence. In some embodiments, the template nucleic acidcomprises at least 100, 150, 200, 300, 400, 500, 600, 700, 800, 900,1000, 1500, or 2000 nucleotides 5′ of the nick or replacement sequence,3′ of the nick or replacement sequence, or both 5′ and 3′ of the nick orreplacement sequence. In some embodiments, the template nucleic acidcomprises at most 100, 150, 200, 300, 400, 500, 600, 700, 800, 900,1000, 1500, or 2000 nucleotides 5′ of the nick or replacement sequence,3′ of the nick or replacement sequence, or both 5′ and 3′ of the nick orreplacement sequence.

In some embodiments, the template nucleic acid is a lentiviral vector,e.g., an DLV (integration deficiency lentivirus). In some embodiments,the template nucleic acid comprises about 500 to 1000 base pairs ofhomology on either side of the replacement sequence and/or the nick. Insome embodiments, the template nucleic acid comprises about 300, 400,500, 600, 700, 800, 900, 1000, 1500, or 2000 base pairs of homology 5′of the nick or replacement sequence, 3′ of the nick or replacementsequence, or both 5′ and 3′ of the nick or replacement sequence. In someembodiments, the template nucleic acid comprises at least 300, 400, 500,600, 700, 800, 900, 1000, 1500, or 2000 base pairs of homology 5′ of thenick or replacement sequence, 3′ of the nick or replacement sequence, orboth 5′ and 3′ of the nick or replacement sequence. In some embodiments,the template nucleic acid comprises no more than 300, 400, 500, 600,700, 800, 900, 1000, 1500, or 2000 base pairs of homology 5′ of the nickor replacement sequence, 3′ of the nick or replacement sequence, or both5′ and 3′ of the nick or replacement sequence.

In one embodiment, the template nucleic acid comprises one or moremutations, e.g., silent mutations, that prevent Cas9 from recognizingand cleaving the template nucleic acid. The template nucleic acid maycomprise, e.g., at least 1, 2, 3, 4, 5, 10, 20, or 30 silent mutationsrelative to the corresponding sequence in the genome of the cell to bealtered. In embodiments, the template nucleic acid comprises at most 2,3, 4, 5, 10, 20, 30, or 50 silent mutations relative to thecorresponding sequence in the genome of the cell to be altered. In oneembodiment, the cDNA comprises one or more mutations, e.g., silentmutations that prevent Cas9 from recognizing and cleaving the templatenucleic acid. The template nucleic acid may comprise, e.g., at least 1,2, 3, 4, 5, 10, 20, or 30 silent mutations relative to the correspondingsequence in the genome of the cell to be altered. In embodiments, thetemplate nucleic acid comprises at most 2, 3, 4, 5, 10, 20, 30, or 50silent mutations relative to the corresponding sequence in the genome ofthe cell to be altered.

In one embodiment, the template nucleic acid alters the structure of thetarget position by participating in a homology directed repair event. Inone embodiment, the template nucleic acid alters the sequence of thetarget position. In one embodiment, the template nucleic acid results inthe incorporation of a modified, or non-naturally occurring base intothe target nucleic acid.

Typically, the template sequence undergoes a breakage mediated orcatalyzed recombination with the target sequence. In one embodiment, thetemplate nucleic acid includes sequence that corresponds to a site onthe target sequence that is cleaved by an eaCas9 mediated cleavageevent. In one embodiment, the template nucleic acid includes sequencethat corresponds to both, a first site on the target sequence that iscleaved in a first Cas9 mediated event, and a second site on the targetsequence that is cleaved in a second Cas9 mediated event.

In one embodiment, the template nucleic acid can include sequence whichresults in an alteration in the coding sequence of a translatedsequence, e.g., one which results in the substitution of one amino acidfor another in a protein product, e.g., transforming a mutant alleleinto a wild type allele, transforming a wild type allele into a mutantallele, and/or introducing a stop codon, insertion of an amino acidresidue, deletion of an amino acid residue, or a nonsense mutation.

In other embodiments, the template nucleic acid can include sequencewhich results in an alteration in a non-coding sequence, e.g., analteration in an exon or in a 5′ or 3′ non-translated or non-transcribedregion. Such alterations include an alteration in a control element,e.g., a promoter, enhancer, and an alteration in a cis-acting ortrans-acting control element.

A template nucleic acid having homology with a target position can beused to alter the structure of a target sequence. The template sequencecan be used to alter an unwanted structure, e.g., an unwanted or mutantnucleotide.

Table 1 below provides exemplary template nucleic acids. In oneembodiment, the template nucleic acid includes the 5′ homology arm andthe 3′ homology arm of a row from Table 1. In another embodiment, a 5′homology arm from the first column can be combined with a 3′ homologyarm from Table 1. In each embodiment, a combination of the 5′ and 3′homology arms include a replacement sequence.

TABLE 1 Length of the 5′ homology Length of the 3′ homology arm (thenumber of Replacement arm (the number of nucleotides) Sequencenucleotides) 10 or more 10 or more 20 or more 20 or more 50 or more 50or more 100 or more 100 or more 150 or more 150 or more 200 or more 200or more 250 or more 250 or more 300 or more 300 or more 350 or more 350or more 400 or more 400 or more 450 or more 450 or more 500 or more 500or more 550 or more 550 or more 600 or more 600 or more 650 or more 650or more 700 or more 700 or more 750 or more 750 or more 800 or more 800or more 850 or more 850 or more 900 or more 900 or more 1000 or more1000 or more 1100 or more 1100 or more 1200 or more 1200 or more 1300 ormore 1300 or more 1400 or more 1400 or more 1500 or more 1500 or more1600 or more 1600 or more 1700 or more 1700 or more 1800 or more 1800 ormore 1900 or more 1900 or more 1200 or more 1200 or more At least 50 butnot long At least 50 but not long enough to include a enough to includea repeated element. repeated element. At least 100 but not long At least100 but not long enough to include a enough to include a repeatedelement. repeated element. At least 150 but not long At least 150 butnot long enough to include a enough to include a repeated element.repeated element. 5 to 100 nucleotides 5 to 100 nucleotides 10 to 150nucleotides 10 to 150 nucleotides 20 to 150 nucleotides 20 to 150nucleotides

“Treat,” “treating” and “treatment,” as used herein, mean the treatmentof a disease in a mammal, e.g., in a human, including (a) inhibiting thedisease, i.e., arresting or preventing its development or progression;(b) relieving the disease, i.e., causing regression of the diseasestate; and (c) relieving one or more symptoms of the disease; and (d)curing the disease.

“Prevent,” “preventing” and “prevention,” as used herein, means theprevention of a disease in a mammal, e.g., in a human, including (a)avoiding or precluding the disease; (b) affecting the predispositiontoward the disease (c) preventing or delaying the onset of at least onesymptom of the disease.

An “up-regulator”, as used herein, refers to an agent that directlyincreases the activity of a specified biological pathway. Directlyincreasing the activity of the pathway refers to (i) the up-regulatorbinding to a component of that pathway (e.g., a protein that acts in thepathway or an mRNA encoding that protein) and increasing the level oractivity of that component, e.g., by increasing the concentration orspecific activity of that component, or (ii) the up-regulator is anadded amount of a component that is ordinarily present in the pathway ata given level, e.g., an overexpressed protein. An up-regulator may,e.g., speed up one of the steps of that pathway or increase the level oractivity of a component in that pathway. An up-regulator may be, e.g., aprotein in the pathway, e.g., one may overexpress a protein that isordinarily in the pathway to increase the overall activity of thepathway. The pathway may be, e.g., a DNA damage repair pathway, forexample, HDR, e.g., gene correction. In one embodiment, the increasedlevel or activity is compared to what would be seen in the absence ofthe up-regulator.

“Wild type”, as used herein, refers to a gene or polypeptide which hasthe characteristics, e.g., the nucleotide or amino acid sequence, of agene or polypeptide from a naturally-occurring source. The term “wildtype” typically includes the most frequent observation of a particulargene or polypeptide in a population of organisms found in nature.

“X” as used herein in the context of an amino acid sequence, refers toany amino acid (e.g., any of the twenty natural amino acids) unlessotherwise specified.

I. Cas9 Fusion Molecules

Various types of Cas9 fusion molecules or Cas9 fusion polypeptides aredisclosed herein. In some embodiments, a Cas9 fusion molecule is achimeric protein comprising a Cas9 protein or a Cas9 polypeptide, or afragment thereof, covalently linked to at least one template nucleicacid. In other embodiments, a Cas9 fusion molecule is a chimeric proteincomprising a Cas9 protein or a Cas9 polypeptide, or a fragment thereof,non-covalently linked to at least one template nucleic acid. ExemplaryCas9 fusion molecules are provided below.

Cas9-Template Nucleic Acid Covalent Fusions

Cas9 molecules of the invention can be directly linked to a templatenucleic acid by various forms of covalent attachment, each of which aredescribed in the subsections, below.

Linkers to Connect Cas9 Molecules to a Template Nucleic Acid

In one embodiment, a linker covalently connects a Cas9 molecule to atemplate nucleic acid to form a Cas9 fusion molecule. A linker may be ashort peptide sequence that connects a protein domain and a nucleic acid(e.g., DNA or RNA). Linkers may be composed of flexible residues likeglycine and serine so that the adjacent protein domains are free to moverelative to one another. In certain embodiments, the linker hassufficient length and flexibility to allow a Cas9 molecule to bind to atarget nucleic acid, e.g., so that the binding event is not stericallyprohibited.

In one embodiment, a linker is attached to the C-terminus of a Cas9molecule. Alternatively, a linker is attached to the N-terminus of aCas9 molecule. In another embodiment, the linker is attached to aposition other than the C-terminus or the N-terminus of a Cas9 molecule,e.g., an internal residue of the Cas9 molecule.

In one embodiment, a linker is attached to the C-terminus of thetemplate nucleic acid. Alternatively, the linker is attached to theN-terminus of the template nucleic acid.

In one embodiment, the linker is attached to the 5′ end of the templatenucleic acid. In another embodiment, the linker is attached to the 3′end of the template nucleic acid. In yet another embodiment, the linkeris attached to a residue other than the 5′-end or the 3′-end of thedonor nucleic acid (e.g., an internal nucleic acid residue).

In some embodiments, the linker from about 3 to 100 amino acids inlength. The linker may be, e.g., 3-10, 6-10, 10-15, 15-20, 20-30, 30-40,40-50, 50-60, 60-70, 70-80, 80-90 or 90-100 amino acids in length. Thelinker may be, e.g., at least 3, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60,70, 80 or 90 amino acids in length. In other embodiments, the linker is,e.g., at most 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90 or 100amino acids in length. Ranges comprising any combination of theseendpoints are also envisioned.

In some embodiments, the linker is encoded by a nucleic acid sequencecomprising about 9 to about 300 nucleotides or base pairs. The nucleicacid may be, e.g., 9-300, 9-210, 9-99, 9-45 nucleotides in length. Thelinker may be, e.g., at least 9, 21, 45, 99, or 210 nucleotides inlength. In some embodiments, the linker is, e.g., at most 9, 18, 21, 45,99, or 210 nucleotides in length. Ranges comprising any combination ofthese endpoints are also envisioned.

In some embodiments, the linker comprises glycine and serine residues.In some embodiments the linker consists of glycine and serine residues.For instance, the linker may comprise one of more modules such as GGS,GSGS, GGGS, GGGGS or GGSG. In some embodiments, the linker comprises aplurality of modules comprising glycine and serine, e.g., at least 2, 3,4, 5, 10, or 15 of these modules, and/or at most 3, 4, 5, 10, 15, or 20of these modules, or any combination of these endpoints. In someembodiments, each module in the linker has the same sequence, and inother embodiments, at least two modules in a linker have differentsequences from each other.

In some embodiments, the linker is an XTEN linker or a variation of anXTEN linker such as SGSETPGTSESA, SGSETPGTSESATPES, orSGSETPGTSESATPEGGSGGS. Additional information on the XTEN linker may befound in Schellenberger et al. (2009), NATURE BIOTECHNOLOGY 27:1186-1190, the entire contents of which are incorporated herein byreference.

Exemplary linker modules are given in Table 2:

Linker SEQ ID NO GGS 206 GSGS 207 GGGS 208 GGGGS 209 GGSG 210SGSETPGTSESA 211 SGSETPGTSESATPES 212 SGSETPGTSESATPEGGSGGS 213GGSGGSGGSGGSGGSGGSGGSGGSGGS 214

Additional exemplary linker modules are given in Table 3:

Length Name Description (nt) BBa_J176131 PLrigid 60 BBa_J18920 2aa GSlinker 6 BBa_J18921 6aa [GS]x linker 18 BBa_J18922 10aa [GS]x linker 30BBa_K105012 10 aa flexible protein domain linker 30 BBa_K133132 8 aaprotein domain linker 24 BBa_K1486003 flexible linker 2x (GGGS) 24BBa_K1486004 flexible linker 2x (GGGGS) 30 BBa_K1486037 linker 39BBa_K157009 Split fluorophore linker; Freiburg 51 standard BBa_K15701315 aa flexible glycine-serine protein 45 domain linker; Freiburgstandard BBa_K243004 Short Linker (Gly-Gly-Ser-Gly) 12 BBa_K243005Middle Linker (Gly-Gly-Ser-Gly)x2 24 BBa_K243006 Long Linker(Gly-Gly-Ser-Gly)x3 36 BBa_K243029 GSAT Linker 108 BBa_K243030 SEG 108BBa_K404300 SEG-Linker 108 BBa_K404301 GSAT-Linker 108 BBa_K404303Z-EGFR-1907_Short-Linker 192 BBa_K404304 Z-EGFR-1907_Middle-Linker 204BBa_K404305 Z-EGFR-1907_Long-Linker 216 BBa_K404306Z-EGFR-1907_SEG-Linker 288 BBa_K416001 (Gly4Ser)3 Flexible PeptideLinker 45 BBa_K648005 Short Fusion Protein Linker: GGSG with 12 standard25 prefix/suffix BBa_K648006 Long 10AA Fusion Protein Linker with 30Standard 25 Prefix/Suffix BBa_K648007 Medium 6AA Fusion Protein Linker:18 GGSGGS with Standard 25 Prefix/Suffix

In another embodiment, linkers can comprise a direct bond or an atomsuch as, e.g., an oxygen (O) or sulfur (S), a unit such as —NR— whereinR is hydrogen or alkyl, —C(O)—, —C(O)O—, —C(O)NH—, SO, SO₂, —SO₂NH— or achain of atoms, such as substituted or unsubstituted alkyl, substitutedor unsubstituted alkenyl, substituted or unsubstituted alkynyl,arylalkyl, heteroarylalkyl. In some embodiments, one or more methylenesin the chain of atoms can be replaced with one or more of O, S, S(O),SO₂, —SO₂NH—, —NR—, —NR₂, —C(O)—, —C(O)O—, —C(O)NH—, a cleavable linkinggroup, substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, and substituted or unsubstituted heterocyclic.

In some embodiments, the template nucleic acid is attached to the Cas9molecule through a linker that is itself stable under physiologicalconditions, such as an alkylene chain, and does not result in release ofthe donor nucleic acid sequence from the Cas9 molecule for at least 2,3, 4, 5, 10, 15, 24 or 48 hours or for at least 1, 2, 3, 4, 5 or 10 dayswhen administered to a subject. In some embodiments, the templatenucleic acid and the Cas9 molecule comprise residues of a functionalgroups through which reaction and linkage of the donor nucleic acidsequence to the Cas9 molecule was achieved. In some embodiments, thefunctional groups, which may be the same or different, terminal orinternal, of the donor nucleic acid sequence or Cas9 molecule comprisean amino, acid, imidazole, hydroxyl, thio, acyl halide, —HC═CH—, —C≡C—group, or derivative thereof. In some embodiments, the linker comprisesa hydrocarbylene group wherein one or more methylene groups isoptionally replaced by a group Y (provided that none of the Y groups areadjacent to each other), wherein each Y, independently for eachoccurrence, is selected from, substituted or unsubstituted aryl,heteroaryl, cycloalkyl, heterocycloalkyl, or —O—, C(═X) (wherein X isNR₁, O or S), —NR₁—, —NR₁C(O)—, —C(O)NR₁—, —S(O)_(n)—, S(O)_(n)NR₁—,—NR₁C(O)—NR₁—; and R₁, independently for each occurrence, represents Hor a lower alkyl and wherein n is 0, 1, or 2.

In some embodiments, the linker comprises an alkylene moiety or aheteroalkylene moiety (e.g., an alkylene glycol moiety such as ethyleneglycol). In some embodiments, a linker comprises a poly-L-glutamic acid,polylactic acid, poly(ethyleneimine), an oligosaccharide, an amino acid(e.g., glycine), an amino acid chain, or any other suitable linkage. Thelinker groups can be biologically inactive, such as a PEG, polyglycolicacid, or polylactic acid chain. In certain embodiments, the linker grouprepresents a derivatized or non-derivatized amino acid (e.g., glycine).

The Cas9 molecule attached to the linker may be any Cas9 moleculedescribed herein, e.g., a nickase Cas9 molecule, or a Cas9 moleculecapable of making a double stranded break.

Direct Cross-Linking Using a Maleimide-Modified Template Nucleic Acid

In one embodiment, a maleimide-modified template nucleic acid iscross-linked to a Cas9 molecule to form a Cas9 fusion molecule.

In one embodiment, template nucleic acid is prepared such that some orall of the sequence contain a maleimide-modification. In someembodiments, template nucleic acid is prepared such that some or all ofthe sequence maleimide-modified at its 5′-end. In one embodiment, thetemplate nucleic acid is single stranded. In an alternate embodiment,the template nucleic acid is double stranded. In one embodiment, thetemplate nucleic acid is DNA, e.g., double stranded DNA. In an alternateembodiment, the template nucleic acid is single stranded DNA.

In one embodiment, the Cas9 molecule is a wild-type molecule. In otherembodiments, the Cas9 molecule is not a wild-type molecule. In certainembodiments, the Cas9 molecule has at least one modification. In anotherembodiment, the Cas9 molecule has at least one modification at asurface-exposed amino acid residue. In other embodiments, the Cas9molecule has at least one mutation. In other embodiments, the Cas9molecule has at least one mutation that results in a change from anon-cysteine amino acid residue to a cysteine amino acid residue. Inother embodiments, the at least one mutation is a mutation forms acysteine-variant Cas9 molecule (e.g., a Cas9 molecule having at leastone cysteine residue). In certain embodiments, the cysteine-variant Cas9molecule has more than one cysteine residue.

In other embodiments, the Cas9 fusion molecule is formed by nature ofthe formation of a covalent bond between the cysteine-variant Cas9molecule (e.g., a Cas9 molecule with at least one surface exposedcysteine residue) and the maleimide-modified template nucleic acid.Without being bound by theory, in some embodiments, it is believed thatthe at least one surface exposed thiol groups on the cysteine-variantCas9 molecule is reactive with the α,β-unsaturated carbonyl component ofthe maleimide-modified template nucleic acid.

The Cas9 molecule attached to the maleimide-modified template nucleicacid may be any Cas9 molecule described herein, e.g., a nickase Cas9molecule or a Cas9 molecule capable of making a double stranded break.

Direct Cross-Linking Via Formation of a Bis-Aryl Hydrazine-BasedConjugate

In one embodiment, a 5′-4-Formylbenzamide (4FB)-modified templatenucleic acid is cross-linked to a 6-hydrazino-nicotinamide(HyNic)-modified Cas9 molecule to form a Cas9 fusion molecule.

In one embodiment, a template nucleic acid is synthesized to have thesome or all of the sequence 4FB-modified. In some embodiments, thetemplate nucleic acid is prepared such that the some or all of thesequence 4FB-modified at its 5′-end. In one embodiment, the templatenucleic acid is single stranded. In an alternate embodiment, thetemplate nucleic acid is double stranded. In one embodiment, thetemplate nucleic acid is DNA, e.g., double stranded DNA. In an alternateembodiment, the template nucleic acid is single stranded DNA.

In one embodiment, the Cas9 molecule is a wild-type molecule. In otherembodiments, the Cas9 molecule is not a wild-type molecule. In certainembodiments, the Cas9 molecule has at least one modification. In anotherembodiment, the Cas9 molecule has at least one modification at asurface-exposed amino acid residue. In another embodiment, the Cas9molecule has at least one modification at a surface-exposed amino acidresidue that is characterized as possessing a primary amine group. Insome embodiments, the at least one modification forms a6-hydrazino-nicotinamide (HyNic)-modified Cas9 molecule. In certainembodiments, the variant Cas9 molecule has more than one6-hydrazino-nicotinamide (HyNic) modification.

In other embodiments, the Cas9 fusion molecule is formed by nature ofthe formation of a covalent bond between the 6-hydrazino-nicotinamide(HyNic)-modified Cas9 molecule and the 4FB-modified template nucleicacid. Without being bound by theory, in some embodiments, it is believedthat a succinimidyl ester functionality of S-HyNic readily reacts withan amine moiety (e.g., a primary amine) on the surface of a Cas9molecule to form a 6-hydrazino-nicotinamide (HyNic)-modified Cas9molecule. Without being bound by theory, in some embodiments, it isbelieved that the at least one surface exposed hydrazino-nicotinamide(HyNic) moiety on the 6-hydrazino-nicotinamide (HyNic)-modified Cas9molecule is reactive with the formylbenzamide (4FB) moiety of the4FB-modified template nucleic acid to form a stable bis-aryl hydrazineconjugate.

The Cas9 molecule attached to the maleimide-modified template nucleicacid may be any Cas9 molecule described herein, e.g., a nickase Cas9molecule or a Cas9 molecule capable of making a double stranded break.

Direct Cross-Linking Using a N-Terminal and/or C-Terminal Cas9 FusionMolecule

Nucleic acid sequences encoding a Cas9 fusion molecule are also providedherein.

In one embodiment, a nucleic acid encoding a Cas9 fusion molecule can bea synthetic nucleic acid sequence. For example, the synthetic nucleicacid molecule can further be chemically modified, e.g., as describedbelow.

In one embodiment, a vector can comprise a nucleic acid sequence thatencodes a Cas9 fusion molecule. In another embodiment, a vector cancomprise a sequence encoding a polypeptide tag (a HaloTag® molecule, aSNAP-Tag®, a CLIP-Tag®, a ACP-Tag® or a MCP-Tag®), fused, e.g., to aCas9 fusion molecule nucleic acid sequence. For example, a vector cancomprise a sequence encoding a polypeptide tag (HaloTag® molecule, aSNAP-Tag®, a CLIP-Tag®, a ACP-Tag® or a MCP-Tag®) fused to the sequenceencoding a Cas9 molecule. In some embodiments, the sequence encoding apolypeptide tag is fused to the N-terminus of the sequence encoding theCas9 molecule. In other embodiments, the sequence encoding a polypeptidetag is fused to the C-terminus of the sequence encoding the Cas9molecule. In certain embodiments, the vector comprises a sequenceencoding a linker between the sequence encoding a polypeptide tag andthe sequence encoding a Cas9 molecule.

In one embodiments, a Cas9 fusion molecule comprises a polypeptide tag(e.g., a HaloTag® molecule, a SNAP-Tag®, a CLIP-Tag®, a ACP-Tag® or aMCP-Tag®) fused to a Cas9 molecule. In certain embodiments, a Cas9fusion molecule comprises a HaloTag fused to the N-terminus of the Cas9molecule. In another embodiment, a Cas9 fusion molecule comprises aHaloTag fused to the C-terminus of the Cas9 molecule. In certainembodiments, a Cas9 fusion molecule comprises a HaloTag fused to alinker sequence (e.g., an XTEN linker, a GGS9 linker, a GGS6 linker, ora GGS linker) fused to the N-terminus of the Cas9 molecule. In anotherembodiment, a Cas9 fusion molecule comprises a HaloTag fused to a linkersequence (e.g., an XTEN linker, a GGS9 linker, a GGS6 linker, or a GGSlinker) fused to the C-terminus of the Cas9 molecule.

In one embodiment, the polypeptide tag is a molecule comprising at leastone modification. In another embodiment, the polypeptide tag has atleast one modification at a surface-exposed amino acid residue. In otherembodiments, the polypeptide tag has at least one mutation. In someembodiments, the at least one mutation comprises a H272F mutation (e.g.,a HaloTag-variant).

In one embodiment, a template nucleic acid is synthesized to have thesome or all of the sequence modified to contain a primary halogen (e.g.,a haloalkane-modified template nucleic acid). In some embodiments, atemplate nucleic acid is synthesized to have the some or all of thesequence modified to contain a primary halogen at its 5′-end. In someembodiments, a template nucleic acid is synthesized to have the some orall of the sequence modified to contain a primary halogen at its 3′-end.In other embodiments, a template nucleic acid is synthesized to have thesome or all of the sequence modified to contain a primary halogen at aninternal position of the template nucleic acid. In one embodiment, thetemplate nucleic acid is single stranded. In an alternate embodiment,the template nucleic acid is double stranded. In one embodiment, thetemplate nucleic acid is DNA, e.g., double stranded DNA. In an alternateembodiment, the template nucleic acid is single stranded DNA.

In other embodiments, the Cas9 fusion molecule is formed by nature ofthe formation of a covalent bond between the polypeptide tag (e.g., aHaloTag-variant) of the Cas9 fusion molecule and the primary halogen ofthe template nucleic acid. Without being bound by theory, in someembodiments, it is believed that certain active site residues of theHaloTag-variant readily reacts via a nucleophilic attack with the atleast one primary halogen on the template nucleic acid to form a stableconjugate.

The Cas9 fusion molecule described herein may be any Cas9 moleculedescribed herein, e.g., a Cas9 nickase molecule, or a Cas9 moleculecapable of making a double stranded break.

Direct Cross-Linking Using an Acrydite-Modified Template Nucleic Acid

In one embodiment, an acrydite-modified template nucleic acid iscross-linked to a Cas9 molecule to form a Cas9 fusion molecule.

In one embodiment, a template nucleic acid is prepared such that some orall of the sequence is acrydite-modified. In some embodiments, templatenucleic acid is prepared such that some or all of the sequence isacrydite-modified at its 5′-end. In some embodiments, template nucleicacid is prepared such that some or all of the sequence isacrydite-modified at its 3′-end. In some embodiments, template nucleicacid is prepared such that some or all of the sequence isacrydite-modified at an internal position of the template nucleic acid.In one embodiment, the template nucleic acid is single stranded. In analternate embodiment, the template nucleic acid is double stranded. Inone embodiment, the template nucleic acid is DNA, e.g., double strandedDNA. In an alternate embodiment, the template nucleic acid is singlestranded DNA.

In one embodiment, the Cas9 molecule is a wild-type molecule. In otherembodiments, the Cas9 molecule is not a wild-type molecule. In certainembodiments, the Cas9 molecule has at least one modification. In anotherembodiment, the Cas9 molecule has at least one modification at asurface-exposed amino acid residue. In other embodiments, the Cas9molecule has at least one mutation. In other embodiments, the Cas9molecule has at least one mutation that results in a change from anon-cysteine amino acid residue to a cysteine amino acid residue. Insome embodiments, the at least one mutation is a mutation that forms acysteine-variant Cas9 molecule (e.g., a Cas9 molecule having at leastone cysteine residue). In certain embodiments, the cysteine-variant Cas9molecule has more than one cysteine residue.

In other embodiments, the Cas9 fusion molecule is formed by nature ofthe formation of a covalent bond between the cysteine-variant Cas9molecule (e.g., a Cas9 molecule with at least one surface exposedcysteine residue) and the acrydite-modified template nucleic acid.Without being bound by theory, in some embodiments, it is believed thatthe at least one surface exposed thiol group (e.g., a surface exposedcysteine) on the cysteine-variant Cas9 molecule is reactive with theacrylic acid moiety of the acrydite-modified template nucleic acid.

The Cas9 molecule attached to the acrydite-modified template nucleicacid may be any Cas9 molecule described herein, e.g., a Cas9 nickasemolecule, or a Cas9 molecule capable of making a double stranded break.

Direct Cross-Linking Using Heterobifunctional Crosslinkers EMCH/EDC

In one embodiment, a carboxy-modified template nucleic acid iscross-linked to a N-[ε-Maleimidocaproic acid] hydrazide (EMCH)-modifiedCas9 molecule via 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide(EDC)-based coupling to form a Cas9 fusion molecule.

In one embodiment, a template nucleic acid is prepared such that some orall of the sequence is carboxy-modified (e.g., a template nucleic acidwith an exposed carboxyl group). In some embodiments, a template nucleicacid is prepared such that some or all of the sequence iscarboxy-modified at its 5′-end. In some embodiments, a template nucleicacid is prepared so that some or all of the sequence carboxy-modified atits 3′-end. In some embodiments, a template nucleic acid is preparedsuch that some or all of the sequence carboxy-modified at an internalposition of the template nucleic acid. In one embodiment, the templatenucleic acid is single stranded. In an alternate embodiment, thetemplate nucleic acid is double stranded. In one embodiment, thetemplate nucleic acid is DNA, e.g., double stranded DNA. In an alternateembodiment, the template nucleic acid is single stranded DNA.

In one embodiment, a carboxy-modified template nucleic acid is coupledto 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) to form anactivated carboxy-modified template nucleic acid (e.g., acarboxy-modified template nucleic acid coupled to EDC) comprising anO-acylisourea ester.

In one embodiment, the Cas9 molecule is a wild-type molecule. In otherembodiments, the Cas9 molecule is not a wild-type molecule. In certainembodiments, the Cas9 molecule has at least one modification. In anotherembodiment, the Cas9 molecule has at least one modification at asurface-exposed amino acid residue. In other embodiments, the Cas9molecule has at least one mutation. In another embodiment, the Cas9molecule has at least one mutation at a surface-exposed amino acidresidue that contains a thiol group. In some embodiments, the at leastone mutation is a mutation that forms a cysteine variant Cas9 molecule(e.g., a Cas9 molecule having at least one cysteine residue). In otherembodiments, the Cas9 molecule has at least one mutation that results ina change from a non-cysteine amino acid residue to a cysteine amino acidresidue. In certain embodiments, the variant Cas9 molecule has more thanone cysteine residue. In some embodiments, the at least one modificationforms a N-[ε-Maleimidocaproic acid] hydrazide (EMCH)-variant Cas9molecule. In certain embodiments, the N-[ε-Maleimidocaproic acid]hydrazide (EMCH)-variant Cas9 molecule has more than one EMCHmodification.

In other embodiments, the Cas9 fusion molecule is formed by nature ofthe formation of a covalent bond between the EMCH-variant Cas9 moleculeand the activated carboxy-modified template nucleic acid (e.g., acarboxy-modified template nucleic acid coupled to EDC). Without beingbound by theory, in some embodiments, it is believed that a primaryamine component of the EMCH-variant Cas9 molecule readily reacts withthe O-acylisourea ester of the activated carboxy-modified templatenucleic acid (e.g., a carboxy-modified template nucleic acid coupled toEDC) to form a stable conjugate.

The Cas9 molecule attached to the maleimide-modified template nucleicacid may be any Cas9 molecule described herein, e.g., a Cas9 nickasemolecule, or a Cas9 molecule capable of making a double stranded break.

Cas9-Donor Template Non-Covalent Fusions

Cas9 fusion molecules described herein may also be linked to a templatenucleic acid by various forms of non-covalent attachment. Non-covalentinteractions generally include hydrogen bonds, ionic bonds, van derWaals interactions, and hydrophobic interactions. Non-covalent linkagesare described in more detail in the subsections, below.

Non-Covalent Attachment Using Biotin and Streptavidin to Form Cas9Fusion Molecules

In one embodiment, a Cas9 molecule is non-covalently attached to atemplate nucleic acid using biotin and streptavidin to form a Cas9fusion molecule.

In one embodiment, a Cas9 molecule is covalently linked to a firstligand. In some embodiments, the first ligand is biotin. In certainembodiments, the Cas9 molecule comprises a linker between the Cas9molecule and the first ligand. In other embodiments, the linker betweenthe Cas9 molecule and the first ligand is sufficiently long to allow theCas9 molecule to bind to a target nucleic acid and the exogenous donortemplate sequence without steric interference. In one embodiment, thelinker comprises a polypeptide. In other embodiments, the polypeptidecomprises serine, glycine, or glycine and serine. In other embodiments,the polypeptide comprises a XTEN-based linker. In some embodiments, thelength of the linker varies from at least 3 amino acid residues to atleast 60 amino acids in length. In one embodiment, the first ligand isan affinity ligand (e.g., a high affinity ligand).

In one embodiment, a template nucleic acid is covalently linked to asecond ligand. In some embodiments, the second ligand is biotin. In oneembodiment, the second ligand is an affinity ligand (e.g., a highaffinity ligand).

In another embodiment, a ligand acceptor protein is bound non-covalentlyand directly to both the first and second affinity ligand (e.g., a highaffinity ligand) at distinct ligand binding sites on a ligand acceptorprotein. In certain embodiments, the ligand acceptor protein isstreptavidin. Without being bound by theory, in some embodiments, it isbelieved that by nature of the ability of the ligand acceptor proteinbinding to a first and a second affinity ligand (e.g., a high affinityligand), a Cas9 molecule is non-covalently linked to a template nucleicacid forming a Cas9 fusion molecule.

The Cas9 molecule non-covalently attached to a template nucleic acid maybe any Cas9 molecule described herein, e.g., a Cas9 nickase molecule, ora Cas9 molecule capable of making a double stranded break.

Non-Covalent Attachment Using Nucleic Acid Binding Proteins to Form Cas9Fusion Molecules

In one embodiment, a Cas9 molecule is non-covalently attached to atemplate nucleic acid using a nucleic acid binding protein to form aCas9 fusion molecule. For example, an eaCas9 molecule may be covalentlylinked to a polypeptide, e.g., a nucleic acid binding protein whereinthe polypeptide is non-covalently bound to the template nucleic acid.

Nucleic acid binding proteins are well known to one of ordinary skill inthe art. For example, nucleic acid binding proteins include, but are notlimited to, Rad52, Rad52-yeast, RPA-4 subunit, BRCA2, Rad51, Rad51B,Rad51C, XRCC2, XRCC3, RecA, RadA, HNRNPA1, UP1 Filament of HNRNPA1,NABP2 (SSB1), NABP1 (SSB2), and UHRF1.

The Cas9 molecule non-covalently attached to a template nucleic acid maybe any Cas9 molecule described herein, e.g., a Cas9 nickase molecule, ora Cas9 molecule capable of making a double stranded break.

Guide RNA (gRNA) Molecules

A gRNA molecule, as that term is used herein, refers to a nucleic acidthat promotes the specific targeting or homing of a gRNA molecule/Cas9molecule complex to a target nucleic acid. gRNA molecules can beunimolecular (having a single RNA molecule) (e.g., chimeric or modular(comprising more than one, and typically two, separate RNA molecules).The gRNA molecules provided herein comprise a targeting domaincomprising, consisting of, or consisting essentially of a nucleic acidsequence fully or partially complementary to a target domain. In certainembodiments, the gRNA molecule further comprises one or more additionaldomains, including for example a first complementarity domain, a linkingdomain, a second complementarity domain, a proximal domain, a taildomain, and a 5′ extension domain. Each of these domains is discussed indetail below. Additional details on gRNAs are provided in Section Ientitled “gRNA molecules” of PCT Application WO 2015/048577, the entirecontents of which are expressly incorporated herein by reference. Incertain embodiments, one or more of the domains in the gRNA moleculecomprises an amino acid sequence identical to or sharing sequencehomology with a naturally occurring sequence, e.g., from S. pyogenes, S.aureus, or S. thermophilus.

In certain embodiments, a unimolecular, or chimeric, gRNA comprises,preferably from 5′ to 3′:

-   -   a targeting domain complementary to a target domain in a target        gene;    -   a first complementarity domain;    -   a linking domain;    -   a second complementarity domain (which is complementary to the        first complementarity domain);    -   a proximal domain; and    -   optionally, a tail domain.

In certain embodiments, a modular gRNA comprises:

-   -   a first strand comprising, preferably from 5′ to 3′:        -   a targeting domain (which is complementary to a target            domain in the target gene); and        -   a first complementarity domain; and    -   a second strand, comprising, preferably from 5′ to 3′:        -   optionally, a 5′ extension domain;        -   a second complementarity domain;        -   a proximal domain; and        -   optionally, a tail domain.

Each of these domains are described in more detail, below.

Targeting Domain

The targeting domain (sometimes referred to alternatively as the guidesequence or complementarity region) comprises, consists of, or consistsessentially of a nucleic acid sequence that is complementary orpartially complementary to a target nucleic acid sequence, e.g., atarget nucleic acid sequence in a target gene. The nucleic acid sequencein a target gene to which all or a portion of the targeting domain iscomplementary or partially complementary is referred to herein as thetarget domain. In certain embodiments, the target domain comprises atarget position within the target gene. In other embodiments, a targetposition lies outside (i.e., upstream or downstream of) the targetdomain. In certain embodiments, the target domain is located entirelywithin a target gene, e.g., in a coding region, an intron, or an exon.In other embodiments, all or part of the target domain is locatedoutside of a target gene, e.g., in a control region or in a non-codingregion.

Methods for selecting targeting domains are known in the art (see, e.g.,Fu 2014; Sternberg 2014).

The strand of the target nucleic acid comprising the target domain isreferred to herein as the “complementary strand” because it iscomplementary to the targeting domain sequence. Since the targetingdomain is part of a gRNA molecule, it comprises the base uracil (U)rather than thymine (T); conversely, any DNA molecule encoding the gRNAmolecule will comprise thymine rather than uracil. In a targetingdomain/target domain pair, the uracil bases in the targeting domain willpair with the adenine bases in the target domain. In certainembodiments, the degree of complementarity between the targeting domainand target domain is sufficient to allow targeting of a Cas9 molecule tothe target nucleic acid.

In certain embodiments, the targeting domain comprises a core domain andan optional secondary domain. In certain of these embodiments, the coredomain is located 3′ to the secondary domain, and in certain of theseembodiments the core domain is located at or near the 3′ end of thetargeting domain. In certain of these embodiments, the core domainconsists of or consists essentially of about 8 to about 13 nucleotidesat the 3′ end of the targeting domain. In certain embodiments, only thecore domain is complementary or partially complementary to thecorresponding portion of the target domain, and in certain of theseembodiments the core domain is fully complementary to the correspondingportion of the target domain. In other embodiments, the secondary domainis also complementary or partially complementary to a portion of thetarget domain. In certain embodiments, the core domain is complementaryor partially complementary to a core domain target in the target domain,while the secondary domain is complementary or partially complementaryto a secondary domain target in the target domain. In certainembodiments, the core domain and secondary domain have the same degreeof complementarity with their respective corresponding portions of thetarget domain. In other embodiments, the degree of complementaritybetween the core domain and its target and the degree of complementaritybetween the secondary domain and its target may differ. In certain ofthese embodiments, the core domain may have a higher degree ofcomplementarity for its target than the secondary domain, whereas inother embodiments the secondary domain may have a higher degree ofcomplementarity than the core domain.

In certain embodiments, the targeting domain and/or the core domainwithin the targeting domain is 3 to 100, 5 to 100, 10 to 100, or 20 to100 nucleotides in length, and in certain of these embodiments thetargeting domain or core domain is 3 to 15, 3 to 20, 5 to 20, 10 to 20,15 to 20, 5 to 50, 10 to 50, or 20 to 50 nucleotides in length. Incertain embodiments, the targeting domain and/or the core domain withinthe targeting domain is 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, or 26 nucleotides in length. In certainembodiments, the targeting domain and/or the core domain within thetargeting domain is 6+/−2, 7+/−2, 8+/−2, 9+/−2, 10+/−2, 10+/−4, 10+/−5,11+/−2, 12+/−2, 13+/−2, 14+/−2, 15+/−2, or 16+−2, 20+/−5, 30+/−5,40+/−5, 50+/−5, 60+/−5, 70+/−5, 80+/−5, 90+/−5, or 100+/−5 nucleotidesin length.

In certain embodiments wherein the targeting domain includes a coredomain, the core domain is 3 to 20 nucleotides in length, and in certainof these embodiments the core domain 5 to 15 or 8 to 13 nucleotides inlength. In certain embodiments wherein the targeting domain includes asecondary domain, the secondary domain is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14 or 15 nucleotides in length. In certain embodimentswherein the targeting domain comprises a core domain that is 8 to 13nucleotides in length, the targeting domain is 26, 25, 24, 23, 22, 21,20, 19, 18, 17, or 16 nucleotides in length, and the secondary domain is13 to 18, 12 to 17, 11 to 16, 10 to 15, 9 to 14, 8 to 13, 7 to 12, 6 to11, 5 to 10, 4 to 9, or 3 to 8 nucleotides in length, respectively.

In certain embodiments, the targeting domain is fully complementary tothe target domain. Likewise, where the targeting domain comprises a coredomain and/or a secondary domain, in certain embodiments one or both ofthe core domain and the secondary domain are fully complementary to thecorresponding portions of the target domain. In other embodiments, thetargeting domain is partially complementary to the target domain, and incertain of these embodiments where the targeting domain comprises a coredomain and/or a secondary domain, one or both of the core domain and thesecondary domain are partially complementary to the correspondingportions of the target domain. In certain of these embodiments, thenucleic acid sequence of the targeting domain, or the core domain ortargeting domain within the targeting domain, is at least 80%, 85%, 90%,or 95% complementary to the target domain or to the correspondingportion of the target domain. In certain embodiments, the targetingdomain and/or the core or secondary domains within the targeting domaininclude one or more nucleotides that are not complementary with thetarget domain or a portion thereof, and in certain of these embodimentsthe targeting domain and/or the core or secondary domains within thetargeting domain include 1, 2, 3, 4, 5, 6, 7, or 8 nucleotides that arenot complementary with the target domain. In certain embodiments, thecore domain includes 1, 2, 3, 4, or 5 nucleotides that are notcomplementary with the corresponding portion of the target domain. Incertain embodiments wherein the targeting domain includes one or morenucleotides that are not complementary with the target domain, one ormore of said non-complementary nucleotides are located within fivenucleotides of the 5′ or 3′ end of the targeting domain. In certain ofthese embodiments, the targeting domain includes 1, 2, 3, 4, or 5nucleotides within five nucleotides of its 5′ end, 3′ end, or both its5′ and 3′ ends that are not complementary to the target domain. Incertain embodiments wherein the targeting domain includes two or morenucleotides that are not complementary to the target domain, two or moreof said non-complementary nucleotides are adjacent to one another, andin certain of these embodiments the two or more consecutivenon-complementary nucleotides are located within five nucleotides of the5′ or 3′ end of the targeting domain. In other embodiments, the two ormore consecutive non-complementary nucleotides are both located morethan five nucleotides from the 5′ and 3′ ends of the targeting domain.

In one embodiment, the gRNA molecule, e.g., a gRNA molecule comprising atargeting domain, which is complementary with the target gene, is amodular gRNA molecule. In another embodiment, the gRNA molecule is aunimolecular or chimeric gRNA molecule.

In one embodiment, the nucleic acid encodes a gRNA molecule, e.g., thefirst gRNA molecule, comprising a targeting domain comprising a sequencethat is the same as, or differs by no more than 1, 2, 3, 4, or 5nucleotides from, a targeting domain sequence described herein. In oneembodiment, the nucleic acid encodes a gRNA molecule comprising atargeting domain described herein.

In certain embodiments, the targeting domain comprises 16 nucleotides.In certain embodiments, the targeting domain comprises 17 nucleotides.In certain embodiments, the targeting domain comprises 18 nucleotides.In certain embodiments, the targeting domain comprises 19 nucleotides.In certain embodiments, the targeting domain comprises 20 nucleotides.In certain embodiments, the targeting domain comprises 21 nucleotides.In certain embodiments, the targeting domain comprises 22 nucleotides.In certain embodiments, the targeting domain comprises 23 nucleotides.In certain embodiments, the targeting domain comprises 24 nucleotides.In certain embodiments, the targeting domain comprises 25 nucleotides.In certain embodiments, the targeting domain comprises 26 nucleotides.

In certain embodiments, the targeting domain which is complementary withthe target gene is 16 nucleotides or more in length. In certainembodiments, the targeting domain is 16 nucleotides in length. Incertain embodiments, the targeting domain is 17 nucleotides in length.In another embodiment, the targeting domain is 18 nucleotides in length.In still another embodiment, the targeting domain is 19 nucleotides inlength. In still another embodiment, the targeting domain is 20nucleotides in length. In still another embodiment, the targeting domainis 21 nucleotides in length. In still another embodiment, the targetingdomain is 22 nucleotides in length. In still another embodiment, thetargeting domain is 23 nucleotides in length. In still anotherembodiment, the targeting domain is 24 nucleotides in length. In stillanother embodiment, the targeting domain is 25 nucleotides in length. Instill another embodiment, the targeting domain is 26 nucleotides inlength.

In one embodiment, a nucleic acid encodes a modular gRNA molecule, e.g.,one or more nucleic acids encode a modular gRNA molecule. In anotherembodiment, a nucleic acid encodes a chimeric gRNA molecule. The nucleicacid may encode a gRNA molecule, e.g., the first gRNA molecule,comprising a targeting domain comprising 16 nucleotides or more inlength. In one embodiment, the nucleic acid encodes a gRNA molecule,e.g., the first gRNA molecule, comprising a targeting domain that is 16nucleotides in length. In another embodiment, the nucleic acid encodes agRNA molecule, e.g., the first gRNA molecule, comprising a targetingdomain that is 17 nucleotides in length. In still another embodiment,the nucleic acid encodes a gRNA molecule, e.g., the first gRNA molecule,comprising a targeting domain that is 18 nucleotides in length. In stillanother embodiment, the nucleic acid encodes a gRNA molecule, e.g., thefirst gRNA molecule, comprising a targeting domain that is 19nucleotides in length. In still another embodiment, the nucleic acidencodes a gRNA molecule, e.g., the first gRNA molecule, comprising atargeting domain that is 20 nucleotides in length. In still anotherembodiment, the nucleic acid encodes a gRNA molecule, e.g., the firstgRNA molecule, comprising a targeting domain that is 21 nucleotides inlength. In still another embodiment, the nucleic acid encodes a gRNAmolecule, e.g., the first gRNA molecule, comprising a targeting domainthat is 22 nucleotides in length. In still another embodiment, thenucleic acid encodes a gRNA molecule, e.g., the first gRNA molecule,comprising a targeting domain that is 23 nucleotides in length. In stillanother embodiment, the nucleic acid encodes a gRNA molecule, e.g., thefirst gRNA molecule, comprising a targeting domain that is 24nucleotides in length. In still another embodiment, the nucleic acidencodes a gRNA molecule, e.g., the first gRNA molecule, comprising atargeting domain that is 25 nucleotides in length. In still anotherembodiment, the nucleic acid encodes a gRNA molecule, e.g., the firstgRNA molecule, comprising a targeting domain that is 26 nucleotides inlength.

In certain embodiments, the targeting domain, core domain, and/orsecondary domain do not comprise any modifications. In otherembodiments, the targeting domain, core domain, and/or secondary domain,or one or more nucleotides therein, have a modification, including butnot limited to the modifications set forth below. In certainembodiments, one or more nucleotides of the targeting domain, coredomain, and/or secondary domain may comprise a 2′ modification (e.g., amodification at the 2′ position on ribose), e.g., a 2-acetylation, e.g.,a 2′ methylation. In certain embodiments, the backbone of the targetingdomain can be modified with a phosphorothioate. In certain embodiments,modifications to one or more nucleotides of the targeting domain, coredomain, and/or secondary domain render the targeting domain and/or thegRNA comprising the targeting domain less susceptible to degradation ormore bio-compatible, e.g., less immunogenic. In certain embodiments, thetargeting domain and/or the core or secondary domains include 1, 2, 3,4, 5, 6, 7, or 8 or more modifications, and in certain of theseembodiments the targeting domain and/or core or secondary domainsinclude 1, 2, 3, or 4 modifications within five nucleotides of theirrespective 5′ ends and/or 1, 2, 3, or 4 modifications within fivenucleotides of their respective 3′ ends. In certain embodiments, thetargeting domain and/or the core or secondary domains comprisemodifications at two or more consecutive nucleotides.

In certain embodiments wherein the targeting domain includes core andsecondary domains, the core and secondary domains contain the samenumber of modifications. In certain of these embodiments, both domainsare free of modifications. In other embodiments, the core domainincludes more modifications than the secondary domain, or vice versa.

In certain embodiments, modifications to one or more nucleotides in thetargeting domain, including in the core or secondary domains, areselected to not interfere with targeting efficacy, which can beevaluated by testing a candidate modification using a system as setforth below. gRNAs having a candidate targeting domain having a selectedlength, sequence, degree of complementarity, or degree of modificationcan be evaluated using a system as set forth below. The candidatetargeting domain can be placed, either alone or with one or more othercandidate changes in a gRNA molecule/Cas9 molecule system known to befunctional with a selected target, and evaluated.

In certain embodiments, all of the modified nucleotides arecomplementary to and capable of hybridizing to corresponding nucleotidespresent in the target domain. In another embodiment, 1, 2, 3, 4, 5, 6, 7or 8 or more modified nucleotides are not complementary to or capable ofhybridizing to corresponding nucleotides present in the target domain.

First and Second Complementarity Domains

The first and second complementarity (sometimes referred toalternatively as the crRNA-derived hairpin sequence and tracrRNA-derivedhairpin sequences, respectively) domains are fully or partiallycomplementary to one another. In certain embodiments, the degree ofcomplementarity is sufficient for the two domains to form a duplexedregion under at least some physiological conditions. In certainembodiments, the degree of complementarity between the first and secondcomplementarity domains, together with other properties of the gRNA, issufficient to allow targeting of a Cas9 molecule to a target nucleicacid.

In certain embodiments the first and/or second complementarity domainincludes one or more nucleotides that lack complementarity with thecorresponding complementarity domain. In certain embodiments, the firstand/or second complementarity domain includes 1, 2, 3, 4, 5, or 6nucleotides that do not complement with the correspondingcomplementarity domain. For example, the second complementarity domainmay contain 1, 2, 3, 4, 5, or 6 nucleotides that do not pair withcorresponding nucleotides in the first complementarity domain. Incertain embodiments, the nucleotides on the first or secondcomplementarity domain that do not complement with the correspondingcomplementarity domain loop out from the duplex formed between the firstand second complementarity domains. In certain of these embodiments, theunpaired loop-out is located on the second complementarity domain, andin certain of these embodiments the unpaired region begins 1, 2, 3, 4,5, or 6 nucleotides from the 5′ end of the second complementaritydomain.

In certain embodiments, the first complementarity domain is 5 to 30, 5to 25, 7 to 25, 5 to 24, 5 to 23, 7 to 22, 5 to 22, 5 to 21, 5 to 20, 7to 18, 7 to 15, 9 to 16, or 10 to 14 nucleotides in length, and incertain of these embodiments the first complementarity domain is 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or25 nucleotides in length. In certain embodiments, the secondcomplementarity domain is 5 to 27, 7 to 27, 7 to 25, 5 to 24, 5 to 23, 5to 22, 5 to 21, 7 to 20, 5 to 20, 7 to 18, 7 to 17, 9 to 16, or 10 to 14nucleotides in length, and in certain of these embodiments the secondcomplementarity domain is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, or 26 nucleotides in length. In certainembodiments, the first and second complementarity domains are eachindependently 6+/−2, 7+/−2, 8+/−2, 9+/−2, 10+/−2, 11+/−2, 12+/−2,13+/−2, 14+/−2, 15+/−2, 16+/−2, 17+/−2, 18+/−2, 19+/−2, or 20+/−2,21+/−2, 22+/−2, 23+/−2, or 24+/−2 nucleotides in length. In certainembodiments, the second complementarity domain is longer than the firstcomplementarity domain, e.g., 2, 3, 4, 5, or 6 nucleotides longer.

In certain embodiments, the first and/or second complementarity domainseach independently comprise three subdomains, which, in the 5′ to 3′direction are: a 5′ subdomain, a central subdomain, and a 3′ subdomain.In certain embodiments, the 5′ subdomain and 3′ subdomain of the firstcomplementarity domain are fully or partially complementary to the 3′subdomain and 5′ subdomain, respectively, of the second complementaritydomain.

In certain embodiments, the 5′ subdomain of the first complementaritydomain is 4 to 9 nucleotides in length, and in certain of theseembodiments the 5′ domain is 4, 5, 6, 7, 8, or 9 nucleotides in length.In certain embodiments, the 5′ subdomain of the second complementaritydomain is 3 to 25, 4 to 22, 4 to 18, or 4 to 10 nucleotides in length,and in certain of these embodiments the 5′ domain is 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25nucleotides in length. In certain embodiments, the central subdomain ofthe first complementarity domain is 1, 2, or 3 nucleotides in length. Incertain embodiments, the central subdomain of the second complementaritydomain is 1, 2, 3, 4, or 5 nucleotides in length. In certainembodiments, the 3′ subdomain of the first complementarity domain is 3to 25, 4 to 22, 4 to 18, or 4 to 10 nucleotides in length, and incertain of these embodiments the 3′ subdomain is 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25nucleotides in length. In certain embodiments, the 3′ subdomain of thesecond complementarity domain is 4 to 9, e.g., 4, 5, 6, 7, 8 or 9nucleotides in length.

The first and/or second complementarity domains can share homology with,or be derived from, naturally occurring or reference first and/or secondcomplementarity domain. In certain of these embodiments, the firstand/or second complementarity domains have at least 50%, 60%, 70%, 80%,85%, 90%, or 95% homology with, or differ by no more than 1, 2, 3, 4, 5,or 6 nucleotides from, the naturally occurring or reference first and/orsecond complementarity domain. In certain of these embodiments, thefirst and/or second complementarity domains may have at least 50%, 60%,70%, 80%, 85%, 90%, or 95% homology with homology with a first and/orsecond complementarity domain from S. pyogenes or S. aureus.

In certain embodiments, the first and/or second complementarity domainsdo not comprise any modifications. In other embodiments, the firstand/or second complementarity domains or one or more nucleotides thereinhave a modification, including but not limited to a modification setforth below. In certain embodiments, one or more nucleotides of thefirst and/or second complementarity domain may comprise a 2′modification (e.g., a modification at the 2′ position on ribose), e.g.,a 2-acetylation, e.g., a 2′ methylation. In certain embodiments, thebackbone of the targeting domain can be modified with aphosphorothioate. In certain embodiments, modifications to one or morenucleotides of the first and/or second complementarity domain render thefirst and/or second complementarity domain and/or the gRNA comprisingthe first and/or second complementarity less susceptible to degradationor more bio-compatible, e.g., less immunogenic. In certain embodiments,the first and/or second complementarity domains each independentlyinclude 1, 2, 3, 4, 5, 6, 7, or 8 or more modifications, and in certainof these embodiments the first and/or second complementarity domainseach independently include 1, 2, 3, or 4 modifications within fivenucleotides of their respective 5′ ends, 3′ ends, or both their 5′ and3′ ends. In other embodiments, the first and/or second complementaritydomains each independently contain no modifications within fivenucleotides of their respective 5′ ends, 3′ ends, or both their 5′ and3′ ends. In certain embodiments, one or both of the first and secondcomplementarity domains comprise modifications at two or moreconsecutive nucleotides.

In certain embodiments, modifications to one or more nucleotides in thefirst and/or second complementarity domains are selected to notinterfere with targeting efficacy, which can be evaluated by testing acandidate modification in a system as set forth below. gRNAs having acandidate first or second complementarity domain having a selectedlength, sequence, degree of complementarity, or degree of modificationcan be evaluated in a system as set forth below. The candidatecomplementarity domain can be placed, either alone or with one or moreother candidate changes in a gRNA molecule/Cas9 molecule system known tobe functional with a selected target, and evaluated.

In certain embodiments, the duplexed region formed by the first andsecond complementarity domains is, for example, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 bp in length, excluding anylooped out or unpaired nucleotides.

In certain embodiments, the first and second complementarity domains,when duplexed, comprise 11 paired nucleotides (see, for e.g., gRNA ofSEQ ID NO:5). In certain embodiments, the first and secondcomplementarity domains, when duplexed, comprise 15 paired nucleotides(see, e.g., gRNA of SEQ ID NO:27). In certain embodiments, the first andsecond complementarity domains, when duplexed, comprise 16 pairednucleotides (see, e.g., gRNA of SEQ ID NO:28). In certain embodiments,the first and second complementarity domains, when duplexed, comprise 21paired nucleotides (see, e.g., gRNA of SEQ ID NO:29).

In certain embodiments, one or more nucleotides are exchanged betweenthe first and second complementarity domains to remove poly-U tracts.For example, nucleotides 23 and 48 or nucleotides 26 and 45 of the gRNAof SEQ ID NO:5 may be exchanged to generate the gRNA of SEQ ID NOs:30 or31, respectively. Similarly, nucleotides 23 and 39 of the gRNA of SEQ IDNO:29 may be exchanged with nucleotides 50 and 68 to generate the gRNAof SEQ ID NO:32.

Linking Domain

The linking domain is disposed between and serves to link the first andsecond complementarity domains in a unimolecular or chimeric gRNA. Incertain embodiments, part of the linking domain is from a crRNA-derivedregion, and another part is from a tracrRNA-derived region.

In certain embodiments, the linking domain links the first and secondcomplementarity domains covalently. In certain of these embodiments, thelinking domain consists of or comprises a covalent bond. In otherembodiments, the linking domain links the first and secondcomplementarity domains non-covalently. In certain embodiments, thelinking domain is ten or fewer nucleotides in length, e.g., 1, 2, 3, 4,5, 6, 7, 8, 9, or 10 nucleotides. In other embodiments, the linkingdomain is greater than 10 nucleotides in length, e.g., 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 or more nucleotides. Incertain embodiments, the linking domain is 2 to 50, 2 to 40, 2 to 30, 2to 20, 2 to 10, 2 to 5, 10 to 100, 10 to 90, 10 to 80, 10 to 70, 10 to60, 10 to 50, 10 to 40, 10 to 30, 10 to 20, 10 to 15, 20 to 100, 20 to90, 20 to 80, 20 to 70, 20 to 60, 20 to 50, 20 to 40, 20 to 30, or 20 to25 nucleotides in length. In certain embodiments, the linking domain is10+/−5, 20+/−5, 20+/−10, 30+/−5, 30+/−10, 40+/−5, 40+/−10, 50+/−5,50+/−10, 60+/−5, 60+/−10, 70+/−5, 70+/−10, 80+/−5, 80+/−10, 90+/−5,90+/−10, 100+/−5, or 100+/−10 nucleotides in length.

In certain embodiments, the linking domain shares homology with, or isderived from, a naturally occurring sequence, e.g., the sequence of atracrRNA that is 5′ to the second complementarity domain. In certainembodiments, the linking domain has at least 50%, 60%, 70%, 80%, 90%, or95% homology with or differs by no more than 1, 2, 3, 4, 5, or 6nucleotides from a linking domain disclosed herein.

In certain embodiments, the linking domain does not comprise anymodifications. In other embodiments, the linking domain or one or morenucleotides therein have a modification, including but not limited tothe modifications set forth below. In certain embodiments, one or morenucleotides of the linking domain may comprise a 2′ modification (e.g.,a modification at the 2′ position on ribose), e.g., a 2-acetylation,e.g., a 2′ methylation. In certain embodiments, the backbone of thelinking domain can be modified with a phosphorothioate. In certainembodiments, modifications to one or more nucleotides of the linkingdomain render the linking domain and/or the gRNA comprising the linkingdomain less susceptible to degradation or more bio-compatible, e.g.,less immunogenic. In certain embodiments, the linking domain includes 1,2, 3, 4, 5, 6, 7, or 8 or more modifications, and in certain of theseembodiments the linking domain includes 1, 2, 3, or 4 modificationswithin five nucleotides of its 5′ and/or 3′ end. In certain embodiments,the linking domain comprises modifications at two or more consecutivenucleotides.

In certain embodiments, modifications to one or more nucleotides in thelinking domain are selected to not interfere with targeting efficacy,which can be evaluated by testing a candidate modification in a systemas set forth below. gRNAs having a candidate linking domain having aselected length, sequence, degree of complementarity, or degree ofmodification can be evaluated in a system as set forth below. Thecandidate linking domain can be placed, either alone or with one or moreother candidate changes in a gRNA molecule/Cas9 molecule system known tobe functional with a selected target, and evaluated.

In certain embodiments, the linking domain comprises a duplexed region,typically adjacent to or within 1, 2, or 3 nucleotides of the 3′ end ofthe first complementarity domain and/or the 5′ end of the secondcomplementarity domain. In certain of these embodiments, the duplexedregion of the linking region is 10+/−5, 15+/−5, 20+/−5, 20+/−10, or30+/−5 bp in length. In certain embodiments, the duplexed region of thelinking domain is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15bp in length. In certain embodiments, the sequences forming the duplexedregion of the linking domain are fully complementarity. In otherembodiments, one or both of the sequences forming the duplexed regioncontain one or more nucleotides (e.g., 1, 2, 3, 4, 5, 6, 7, or 8nucleotides) that are not complementary with the other duplex sequence.

5′ Extension Domain

In certain embodiments, a modular gRNA as disclosed herein comprises a5′ extension domain, i.e., one or more additional nucleotides 5′ to thesecond complementarity domain. In certain embodiments, the 5′ extensiondomain is 2 to 10 or more, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, or 2to 4 nucleotides in length, and in certain of these embodiments the 5′extension domain is 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nucleotides inlength.

In certain embodiments, the 5′ extension domain nucleotides do notcomprise modifications, e.g., modifications of the type provided below.However, in certain embodiments, the 5′ extension domain comprises oneor more modifications, e.g., modifications that it render it lesssusceptible to degradation or more bio-compatible, e.g., lessimmunogenic. By way of example, the backbone of the 5′ extension domaincan be modified with a phosphorothioate, or other modification(s) as setforth below. In certain embodiments, a nucleotide of the 5′ extensiondomain can comprise a 2′ modification (e.g., a modification at the 2′position on ribose), e.g., a 2-acetylation, e.g., a 2′ methylation, orother modification(s) as set forth below.

In certain embodiments, the 5′ extension domain can comprise as many as1, 2, 3, 4, 5, 6, 7, or 8 modifications. In certain embodiments, the 5′extension domain comprises as many as 1, 2, 3, or 4 modifications within5 nucleotides of its 5′ end, e.g., in a modular gRNA molecule. Incertain embodiments, the 5′ extension domain comprises as many as 1, 2,3, or 4 modifications within 5 nucleotides of its 3′ end, e.g., in amodular gRNA molecule.

In certain embodiments, the 5′ extension domain comprises modificationsat two consecutive nucleotides, e.g., two consecutive nucleotides thatare within 5 nucleotides of the 5′ end of the 5′ extension domain,within 5 nucleotides of the 3′ end of the 5′ extension domain, or morethan 5 nucleotides away from one or both ends of the 5′ extensiondomain. In certain embodiments, no two consecutive nucleotides aremodified within 5 nucleotides of the 5′ end of the 5′ extension domain,within 5 nucleotides of the 3′ end of the 5′ extension domain, or withina region that is more than 5 nucleotides away from one or both ends ofthe 5′ extension domain. In certain embodiments, no nucleotide ismodified within 5 nucleotides of the 5′ end of the 5′ extension domain,within 5 nucleotides of the 3′ end of the 5′ extension domain, or withina region that is more than 5 nucleotides away from one or both ends ofthe 5′ extension domain.

Modifications in the 5′ extension domain can be selected so as to notinterfere with gRNA molecule efficacy, which can be evaluated by testinga candidate modification in a system as set forth below. gRNAs having acandidate 5′ extension domain having a selected length, sequence, degreeof complementarity, or degree of modification, can be evaluated in asystem as set forth below. The candidate 5′ extension domain can beplaced, either alone, or with one or more other candidate changes in agRNA molecule/Cas9 molecule system known to be functional with aselected target and evaluated.

In certain embodiments, the 5′ extension domain has at least 60, 70, 80,85, 90, or 95% homology with, or differs by no more than 1, 2, 3, 4, 5,or 6 nucleotides from, a reference 5′ extension domain, e.g., anaturally occurring, e.g., an S. pyogenes, S. aureus, or S.thermophilus, 5′ extension domain, or a 5′ extension domain describedherein.

Proximal Domain

In certain embodiments, the proximal domain is 5 to 20 or morenucleotides in length, e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 nucleotides in length. Incertain of these embodiments, the proximal domain is 6+/−2, 7+/−2,8+/−2, 9+/−2, 10+/−2, 11+/−2, 12+/−2, 13+/−2, 14+/−2, 14+/−2, 16+/−2,17+/−2, 18+/−2, 19+/−2, or 20+/−2 nucleotides in length. In certainembodiments, the proximal domain is 5 to 20, 7, to 18, 9 to 16, or 10 to14 nucleotides in length.

In certain embodiments, the proximal domain can share homology with orbe derived from a naturally occurring proximal domain. In certain ofthese embodiments, the proximal domain has at least 50%, 60%, 70%, 80%,85%, 90%, or 95% homology with or differs by no more than 1, 2, 3, 4, 5,or 6 nucleotides from a proximal domain disclosed herein, e.g., an S.pyogenes, S. aureus, or S. thermophilus proximal domain.

In certain embodiments, the proximal domain does not comprise anymodifications. In other embodiments, the proximal domain or one or morenucleotides therein have a modification, including but not limited tothe modifications set forth in herein. In certain embodiments, one ormore nucleotides of the proximal domain may comprise a 2′ modification(e.g., a modification at the 2′ position on ribose), e.g., a2-acetylation, e.g., a 2′ methylation. In certain embodiments, thebackbone of the proximal domain can be modified with a phosphorothioate.In certain embodiments, modifications to one or more nucleotides of theproximal domain render the proximal domain and/or the gRNA comprisingthe proximal domain less susceptible to degradation or morebio-compatible, e.g., less immunogenic. In certain embodiments, theproximal domain includes 1, 2, 3, 4, 5, 6, 7, or 8 or moremodifications, and in certain of these embodiments the proximal domainincludes 1, 2, 3, or 4 modifications within five nucleotides of its 5′and/or 3′ end. In certain embodiments, the proximal domain comprisesmodifications at two or more consecutive nucleotides.

In certain embodiments, modifications to one or more nucleotides in theproximal domain are selected to not interfere with targeting efficacy,which can be evaluated by testing a candidate modification in a systemas set forth below. gRNAs having a candidate proximal domain having aselected length, sequence, degree of complementarity, or degree ofmodification can be evaluated in a system as set forth below. Thecandidate proximal domain can be placed, either alone or with one ormore other candidate changes in a gRNA molecule/Cas9 molecule systemknown to be functional with a selected target, and evaluated.

Tail Domain

A broad spectrum of tail domains are suitable for use in the gRNAmolecules disclosed herein.

In certain embodiments, the tail domain is absent. In other embodiments,the tail domain is 1 to 100 or more nucleotides in length, e.g., 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100nucleotides in length. In certain embodiments, the tail domain is 1 to5, 1 to 10, 1 to 15, 1 to 20, 1 to 50, 10 to 100, 20 to 100, 10 to 90,20 to 90, 10 to 80, 20 to 80, 10 to 70, 20 to 70, 10 to 60, 20 to 60, 10to 50, 20 to 50, 10 to 40, 20 to 40, 10 to 30, 20 to 30, 20 to 25, 10 to20, or 10 to 15 nucleotides in length. In certain embodiments, the taildomain is 5+/−5, 10+/−5, 20+/−10, 20+/−5, 25+/−10, 30+/−10, 30+/−5,40+/−10, 40+/−5, 50+/−10, 50+/−5, 60+/−10, 60+/−5, 70+/−10, 70+/−5,80+/−10, 80+/−5, 90+/−10, 90+/−5, 100+/−10, or 100+/−5 nucleotides inlength,

In certain embodiments, the tail domain can share homology with or bederived from a naturally occurring tail domain or the 5′ end of anaturally occurring tail domain. In certain of these embodiments, theproximal domain has at least 50%, 60%, 70%, 80%, 85%, 90%, or 95%homology with or differs by no more than 1, 2, 3, 4, 5, or 6 nucleotidesfrom a naturally occurring tail domain disclosed herein, e.g., an S.pyogenes, S. aureus, or S. thermophilus tail domain.

In certain embodiments, the tail domain includes sequences that arecomplementary to each other and which, under at least some physiologicalconditions, form a duplexed region. In certain of these embodiments, thetail domain comprises a tail duplex domain which can form a tailduplexed region. In certain embodiments, the tail duplexed region is 3,4, 5, 6, 7, 8, 9, 10, 11, or 12 bp in length. In certain embodiments,the tail domain comprises a single stranded domain 3′ to the tail duplexdomain that does not form a duplex. In certain of these embodiments, thesingle stranded domain is 3 to 10 nucleotides in length, e.g., 3, 4, 5,6, 7, 8, 9, 10, or 4 to 6 nucleotides in length.

In certain embodiments, the tail domain does not comprise anymodifications. In other embodiments, the tail domain or one or morenucleotides therein have a modification, including but not limited tothe modifications set forth herein. In certain embodiments, one or morenucleotides of the tail domain may comprise a 2′ modification (e.g., amodification at the 2′ position on ribose), e.g., a 2-acetylation, e.g.,a 2′ methylation. In certain embodiments, the backbone of the taildomain can be modified with a phosphorothioate. In certain embodiments,modifications to one or more nucleotides of the tail domain render thetail domain and/or the gRNA comprising the tail domain less susceptibleto degradation or more bio-compatible, e.g., less immunogenic. Incertain embodiments, the tail domain includes 1, 2, 3, 4, 5, 6, 7, or 8or more modifications, and in certain of these embodiments the taildomain includes 1, 2, 3, or 4 modifications within five nucleotides ofits 5′ and/or 3′ end. In certain embodiments, the tail domain comprisesmodifications at two or more consecutive nucleotides.

In certain embodiments, modifications to one or more nucleotides in thetail domain are selected to not interfere with targeting efficacy, whichcan be evaluated by testing a candidate modification as set forth below.gRNAs having a candidate tail domain having a selected length, sequence,degree of complementarity, or degree of modification can be evaluatedusing a system as set forth below. The candidate tail domain can beplaced, either alone or with one or more other candidate changes in agRNA molecule/Cas9 molecule system known to be functional with aselected target, and evaluated.

In certain embodiments, the tail domain includes nucleotides at the 3′end that are related to the method of in vitro or in vivo transcription.When a T7 promoter is used for in vitro transcription of the gRNA, thesenucleotides may be any nucleotides present before the 3′ end of the DNAtemplate. When a U6 promoter is used for in vivo transcription, thesenucleotides may be the sequence UUUUUU. When an H1 promoter is used fortranscription, these nucleotides may be the sequence UUUU. Whenalternate pol-III promoters are used, these nucleotides may be variousnumbers of uracil bases depending on, e.g., the termination signal ofthe pol-III promoter, or they may include alternate bases.

In certain embodiments, the proximal and tail domain taken togethercomprise, consist of, or consist essentially of the sequence set forthin SEQ ID NOs: 33, 34, 35, 36, or 38.

Exemplary Unimolecular/Chimeric gRNAs

In certain embodiments, a gRNA as disclosed herein has the structure: 5′[targeting domain]-[first complementarity domain]-[linkingdomain]-[second complementarity domain]-[proximal domain]-[taildomain]-3′, wherein:

the targeting domain comprises a core domain and optionally a secondarydomain, and is 10 to 50 nucleotides in length;

the first complementarity domain is 5 to 25 nucleotides in length and,in certain embodiments has at least 50, 60, 70, 80, 85, 90, or 95%homology with a reference first complementarity domain disclosed herein;

the linking domain is 1 to 5 nucleotides in length;

the second complementarity domain is 5 to 27 nucleotides in length and,in certain embodiments has at least 50, 60, 70, 80, 85, 90, or 95%homology with a reference second complementarity domain disclosedherein;

the proximal domain is 5 to 20 nucleotides in length and, in certainembodiments has at least 50, 60, 70, 80, 85, 90, or 95% homology with areference proximal domain disclosed herein; and

the tail domain is absent or a nucleotide sequence is 1 to 50nucleotides in length and, in certain embodiments has at least 50, 60,70, 80, 85, 90, or 95% homology with a reference tail domain disclosedherein.

In certain embodiments, a unimolecular gRNA as disclosed hereincomprises, preferably from 5′ to 3′:

-   -   a targeting domain, e.g., comprising 10-50 nucleotides;    -   a first complementarity domain, e.g., comprising 15, 16, 17, 18,        19, 20, 21, 22, 23, 24, 25, or 26 nucleotides;    -   a linking domain;    -   a second complementarity domain;    -   a proximal domain; and    -   a tail domain,

wherein,

-   -   (a) the proximal and tail domain, when taken together, comprise        at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53        nucleotides;    -   (b) there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49,        50, or 53 nucleotides 3′ to the last nucleotide of the second        complementarity domain; or    -   (c) there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50,        51, or 54 nucleotides 3′ to the last nucleotide of the second        complementarity domain that is complementary to its        corresponding nucleotide of the first complementarity domain.

In certain embodiments, the sequence from (a), (b), and/or (c) has atleast 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% homology with thecorresponding sequence of a naturally occurring gRNA, or with a gRNAdescribed herein.

In certain embodiments, the proximal and tail domain, when takentogether, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50,or 53 nucleotides.

In certain embodiments, there are at least 15, 18, 20, 25, 30, 31, 35,40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of thesecond complementarity domain.

In certain embodiments, there are at least 16, 19, 21, 26, 31, 32, 36,41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of thesecond complementarity domain that are complementary to thecorresponding nucleotides of the first complementarity domain.

In certain embodiments, the targeting domain consists of, consistsessentially of, or comprises 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or26 nucleotides (e.g., 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26consecutive nucleotides) complementary or partially complementary to thetarget domain or a portion thereof, e.g., the targeting domain is 16,17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 nucleotides in length. Incertain of these embodiments, the targeting domain is complementary tothe target domain over the entire length of the targeting domain, theentire length of the target domain, or both.

In certain embodiments, a unimolecular or chimeric gRNA moleculedisclosed herein (comprising a targeting domain, a first complementarydomain, a linking domain, a second complementary domain, a proximaldomain and, optionally, a tail domain) comprises the amino acid sequenceset forth in SEQ ID NO:45, wherein the targeting domain is listed as 20N's (residues 1-20) but may range in length from 16 to 26 nucleotides,and wherein the final six residues (residues 97-102) represent atermination signal for the U6 promoter buy may be absent or fewer innumber. In certain embodiments, the unimolecular, or chimeric, gRNAmolecule is a S. pyogenes gRNA molecule.

In certain embodiments, a unimolecular or chimeric gRNA moleculedisclosed herein (comprising a targeting domain, a first complementarydomain, a linking domain, a second complementary domain, a proximaldomain and, optionally, a tail domain) comprises the amino acid sequenceset forth in SEQ ID NO:40, wherein the targeting domain is listed as 20Ns (residues 1-20) but may range in length from 16 to 26 nucleotides,and wherein the final six residues (residues 97-102) represent atermination signal for the U6 promoter but may be absent or fewer innumber. In certain embodiments, the unimolecular or chimeric gRNAmolecule is an S. aureus gRNA molecule.

Exemplary Modular gRNAs

In certain embodiments, a modular gRNA disclosed herein comprises:

-   -   a first strand comprising, preferably from 5′ to 3′;        -   a targeting domain, e.g., comprising 15, 16, 17, 18, 19, 20,            21, 22, 23, 24, 25, or 26 nucleotides;        -   a first complementarity domain; and    -   a second strand, comprising, preferably from 5′ to 3′:        -   optionally a 5′ extension domain;        -   a second complementarity domain;        -   a proximal domain; and        -   a tail domain,

wherein:

-   -   (a) the proximal and tail domain, when taken together, comprise        at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53        nucleotides;    -   (b) there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49,        50, or 53 nucleotides 3′ to the last nucleotide of the second        complementarity domain; or    -   (c) there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50,        51, or 54 nucleotides 3′ to the last nucleotide of the second        complementarity domain that is complementary to its        corresponding nucleotide of the first complementarity domain.

In certain embodiments, the sequence from (a), (b), or (c), has at least60, 75, 80, 85, 90, 95, or 99% homology with the corresponding sequenceof a naturally occurring gRNA, or with a gRNA described herein.

In certain embodiments, the proximal and tail domain, when takentogether, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50,or 53 nucleotides.

In certain embodiments, there are at least 15, 18, 20, 25, 30, 31, 35,40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of thesecond complementarity domain.

In certain embodiments, there are at least 16, 19, 21, 26, 31, 32, 36,41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of thesecond complementarity domain that is complementary to its correspondingnucleotide of the first complementarity domain. In certain embodiments,the targeting domain comprises, has, or consists of, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, or 26 nucleotides (e.g., 16, 17, 18, 19, 20, 21, 22,23, 24, 25, or 26 consecutive nucleotides) having complementarity withthe target domain, e.g., the targeting domain is 16, 17, 18, 19, 20, 21,22, 23, 24, 25, or 26 nucleotides in length.

In certain embodiments, the targeting domain consists of, consistsessentially of, or comprises 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or26 nucleotides (e.g., 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26consecutive nucleotides) complementary to the target domain or a portionthereof. In certain of these embodiments, the targeting domain iscomplementary to the target domain over the entire length of thetargeting domain, the entire length of the target domain, or both.

In certain embodiments, the targeting domain comprises, has, or consistsof, 16 nucleotides (e.g., 16 consecutive nucleotides) havingcomplementarity with the target domain, e.g., the targeting domain is 16nucleotides in length; and the proximal and tail domain, when takentogether, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50,or 53 nucleotides.

In certain embodiments, the targeting domain comprises, has, or consistsof, 16 nucleotides (e.g., 16 consecutive nucleotides) havingcomplementarity with the target domain, e.g., the targeting domain is 16nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31,35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of thesecond complementarity domain.

In certain embodiments, the targeting domain comprises, has, or consistsof, 16 nucleotides (e.g., 16 consecutive nucleotides) havingcomplementarity with the target domain, e.g., the targeting domain is 16nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32,36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of thesecond complementarity domain that is complementary to its correspondingnucleotide of the first complementarity domain.

In certain embodiments, the targeting domain has, or consists of, 17nucleotides (e.g., 17 consecutive nucleotides) having complementaritywith the target domain, e.g., the targeting domain is 17 nucleotides inlength; and the proximal and tail domain, when taken together, compriseat least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

In certain embodiments, the targeting domain has, or consists of, 17nucleotides (e.g., 17 consecutive nucleotides) having complementaritywith the target domain, e.g., the targeting domain is 17 nucleotides inlength; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49,50, or 53 nucleotides 3′ to the last nucleotide of the secondcomplementarity domain.

In certain embodiments, the targeting domain has, or consists of, 17nucleotides (e.g., 17 consecutive nucleotides) having complementaritywith the target domain, e.g., the targeting domain is 17 nucleotides inlength; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50,51, or 54 nucleotides 3′ to the last nucleotide of the secondcomplementarity domain that is complementary to its correspondingnucleotide of the first complementarity domain.

In certain embodiments, the targeting domain has, or consists of, 18nucleotides (e.g., 18 consecutive nucleotides) having complementaritywith the target domain, e.g., the targeting domain is 18 nucleotides inlength; and the proximal and tail domain, when taken together, compriseat least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

In certain embodiments, the targeting domain has, or consists of, 18nucleotides (e.g., 18 consecutive nucleotides) having complementaritywith the target domain, e.g., the targeting domain is 18 nucleotides inlength; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49,50, or 53 nucleotides 3′ to the last nucleotide of the secondcomplementarity domain.

In certain embodiments, the targeting domain has, or consists of, 18nucleotides (e.g., 18 consecutive nucleotides) having complementaritywith the target domain, e.g., the targeting domain is 18 nucleotides inlength; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50,51, or 54 nucleotides 3′ to the last nucleotide of the secondcomplementarity domain that is complementary to its correspondingnucleotide of the first complementarity domain.

In certain embodiments, the targeting domain comprises, has, or consistsof, 19 nucleotides (e.g., 19 consecutive nucleotides) havingcomplementarity with the target domain, e.g., the targeting domain is 19nucleotides in length; and the proximal and tail domain, when takentogether, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50,or 53 nucleotides.

In certain embodiments, the targeting domain comprises, has, or consistsof, 19 nucleotides (e.g., 19 consecutive nucleotides) havingcomplementarity with the target domain, e.g., the targeting domain is 19nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31,35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of thesecond complementarity domain.

In certain embodiments, the targeting domain comprises, has, or consistsof, 19 nucleotides (e.g., 19 consecutive nucleotides) havingcomplementarity with the target domain, e.g., the targeting domain is 19nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32,36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of thesecond complementarity domain that is complementary to its correspondingnucleotide of the first complementarity domain.

In certain embodiments, the targeting domain comprises, has, or consistsof, 20 nucleotides (e.g., 20 consecutive nucleotides) havingcomplementarity with the target domain, e.g., the targeting domain is 20nucleotides in length; and the proximal and tail domain, when takentogether, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50,or 53 nucleotides.

In certain embodiments, the targeting domain comprises, has, or consistsof, 20 nucleotides (e.g., 20 consecutive nucleotides) havingcomplementarity with the target domain, e.g., the targeting domain is 20nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31,35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of thesecond complementarity domain.

In certain embodiments, the targeting domain comprises, has, or consistsof, 20 nucleotides (e.g., 20 consecutive nucleotides) havingcomplementarity with the target domain, e.g., the targeting domain is 20nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32,36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of thesecond complementarity domain that is complementary to its correspondingnucleotide of the first complementarity domain.

In certain embodiments, the targeting domain comprises, has, or consistsof, 21 nucleotides (e.g., 21 consecutive nucleotides) havingcomplementarity with the target domain, e.g., the targeting domain is 21nucleotides in length; and the proximal and tail domain, when takentogether, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50,or 53 nucleotides.

In certain embodiments, the targeting domain comprises, has, or consistsof, 21 nucleotides (e.g., 21 consecutive nucleotides) havingcomplementarity with the target domain, e.g., the targeting domain is 21nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31,35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of thesecond complementarity domain.

In certain embodiments, the targeting domain comprises, has, or consistsof, 21 nucleotides (e.g., 21 consecutive nucleotides) havingcomplementarity with the target domain, e.g., the targeting domain is 21nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32,36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of thesecond complementarity domain that is complementary to its correspondingnucleotide of the first complementarity domain.

In certain embodiments, the targeting domain comprises, has, or consistsof, 22 nucleotides (e.g., 22 consecutive nucleotides) havingcomplementarity with the target domain, e.g., the targeting domain is 22nucleotides in length; and the proximal and tail domain, when takentogether, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50,or 53 nucleotides.

In certain embodiments, the targeting domain comprises, has, or consistsof, 22 nucleotides (e.g., 22 consecutive nucleotides) havingcomplementarity with the target domain, e.g., the targeting domain is 22nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31,35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of thesecond complementarity domain.

In certain embodiments, the targeting domain comprises, has, or consistsof, 22 nucleotides (e.g., 22 consecutive nucleotides) havingcomplementarity with the target domain, e.g., the targeting domain is 22nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32,36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of thesecond complementarity domain that is complementary to its correspondingnucleotide of the first complementarity domain.

In certain embodiments, the targeting domain comprises, has, or consistsof, 23 nucleotides (e.g., 23 consecutive nucleotides) havingcomplementarity with the target domain, e.g., the targeting domain is 23nucleotides in length; and the proximal and tail domain, when takentogether, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50,or 53 nucleotides.

In certain embodiments, the targeting domain comprises, has, or consistsof, 23 nucleotides (e.g., 23 consecutive nucleotides) havingcomplementarity with the target domain, e.g., the targeting domain is 23nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31,35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of thesecond complementarity domain.

In certain embodiments, the targeting domain comprises, has, or consistsof, 23 nucleotides (e.g., 23 consecutive nucleotides) havingcomplementarity with the target domain, e.g., the targeting domain is 23nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32,36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of thesecond complementarity domain that is complementary to its correspondingnucleotide of the first complementarity domain.

In certain embodiments, the targeting domain comprises, has, or consistsof, 24 nucleotides (e.g., 24 consecutive nucleotides) havingcomplementarity with the target domain, e.g., the targeting domain is 24nucleotides in length; and the proximal and tail domain, when takentogether, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50,or 53 nucleotides.

In certain embodiments, the targeting domain comprises, has, or consistsof, 24 nucleotides (e.g., 24 consecutive nucleotides) havingcomplementarity with the target domain, e.g., the targeting domain is 24nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31,35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of thesecond complementarity domain.

In certain embodiments, the targeting domain comprises, has, or consistsof, 24 nucleotides (e.g., 24 consecutive nucleotides) havingcomplementarity with the target domain, e.g., the targeting domain is 24nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32,36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of thesecond complementarity domain that is complementary to its correspondingnucleotide of the first complementarity domain.

In certain embodiments, the targeting domain comprises, has, or consistsof, 25 nucleotides (e.g., 25 consecutive nucleotides) havingcomplementarity with the target domain, e.g., the targeting domain is 25nucleotides in length; and the proximal and tail domain, when takentogether, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50,or 53 nucleotides.

In certain embodiments, the targeting domain comprises, has, or consistsof, 25 nucleotides (e.g., 25 consecutive nucleotides) havingcomplementarity with the target domain, e.g., the targeting domain is 25nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31,35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of thesecond complementarity domain.

In certain embodiments, the targeting domain comprises, has, or consistsof, 25 nucleotides (e.g., 25 consecutive nucleotides) havingcomplementarity with the target domain, e.g., the targeting domain is 25nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32,36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of thesecond complementarity domain that is complementary to its correspondingnucleotide of the first complementarity domain.

In certain embodiments, the targeting domain comprises, has, or consistsof, 26 nucleotides (e.g., 26 consecutive nucleotides) havingcomplementarity with the target domain, e.g., the targeting domain is 26nucleotides in length; and the proximal and tail domain, when takentogether, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50,or 53 nucleotides.

In certain embodiments, the targeting domain comprises, has, or consistsof, 26 nucleotides (e.g., 26 consecutive nucleotides) havingcomplementarity with the target domain, e.g., the targeting domain is 26nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31,35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of thesecond complementarity domain.

In certain embodiments, the targeting domain comprises, has, or consistsof, 26 nucleotides (e.g., 26 consecutive nucleotides) havingcomplementarity with the target domain, e.g., the targeting domain is 26nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32,36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of thesecond complementarity domain that is complementary to its correspondingnucleotide of the first complementarity domain.

Methods for Designing gRNA Molecules Methods for selecting, designing,and validating targeting domains for use in the gRNAs described hereinare provided. Exemplary targeting domains for incorporation into gRNAsare also provided herein.

Methods for selection and validation of target sequences as well asoff-target analyses have been described (see, e.g., Mali 2013; Hsu 2013;Fu 2014; Heigwer 2014; Bae 2014; and Xiao 2014). For example, a softwaretool can be used to optimize the choice of potential targeting domainscorresponding to a user's target sequence, e.g., to minimize totaloff-target activity across the genome. Off-target activity may be otherthan cleavage. For each possible targeting domain choice using S.pyogenes Cas9, the tool can identify all off-target sequences (precedingeither NAG or NGG PAMs) across the genome that contain up to certainnumber (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) of mismatchedbase-pairs. The cleavage efficiency at each off-target sequence can bepredicted, e.g., using an experimentally-derived weighting scheme. Eachpossible targeting domain is then ranked according to its totalpredicted off-target cleavage; the top-ranked targeting domainsrepresent those that are likely to have the greatest on-target cleavageand the least off-target cleavage. Other functions, e.g., automatedreagent design for CRISPR construction, primer design for the on-targetSurveyor assay, and primer design for high-throughput detection andquantification of off-target cleavage via next-gen sequencing, can alsobe included in the tool. Candidate targeting domains and gRNAscomprising those targeting domains can be functionally evaluated byusing methods known in the art and/or as set forth herein.

As a non-limiting example, targeting domains for use in gRNAs for usewith S. pyogenes, and S. aureus Cas9s were identified using a DNAsequence searching algorithm. 17-mer and 20-mer targeting domains weredesigned for S. pyogenes targets, while 18-mer, 19-mer, 20-mer, 21-mer,22-mer, 23-mer, and 24-mer targeting domains were designed for S. aureustargets. gRNA design was carried out using a custom gRNA design softwarebased on the public tool cas-offinder (Bae 2014). This software scoresguides after calculating their genome-wide off-target propensity.Typically matches ranging from perfect matches to 7 mismatches areconsidered for guides ranging in length from 17 to 24. Once theoff-target sites are computationally-determined, an aggregate score iscalculated for each guide and summarized in a tabular output using aweb-interface. In addition to identifying potential target sitesadjacent to PAM sequences, the software also identifies all PAM adjacentsequences that differ by 1, 2, 3 or more than 3 nucleotides from theselected target sites. Genomic DNA sequences for a target gene may beobtained from the UCSC Genome browser and sequences screened for repeatelements using the publically available RepeatMasker program.RepeatMasker searches input DNA sequences for repeated elements andregions of low complexity. The output is a detailed annotation of therepeats present in a given query sequence.

Following identification, targeting domains were ranked into tiers basedon their distance to the target site, their orthogonality and presenceof a 5′ G (based on identification of close matches in the human genomecontaining a relevant PAM e.g., NGG PAM for S. pyogenes, NNGRRT orNNGRRV PAM for S. aureus. Orthogonality refers to the number ofsequences in the human genome that contain a minimum number ofmismatches to the target sequence. A “high level of orthogonality” or“good orthogonality” may, for example, refer to 20-mer targeting domainsthat have no identical sequences in the human genome besides theintended target, nor any sequences that contain one or two mismatches inthe target sequence. Targeting domains with good orthogonality areselected to minimize off-target DNA cleavage.

Targeting domains were identified for both single-gRNA nuclease cleavageand for a dual-gRNA paired “nickase” strategy. Criteria for selectingtargeting domains and the determination of which targeting domains canbe incorporated into a gRNA and used for the dual-gRNA paired “nickase”strategy is based on two considerations:

-   -   1. gRNA pairs should be oriented on the DNA such that PAMs are        facing out and cutting with the D10A Cas9 nickase will result in        5′ overhangs.    -   2. An assumption that cleaving with dual nickase pairs will        result in deletion of the entire intervening sequence at a        reasonable frequency. However, cleaving with dual nickase pairs        can also result in indel mutations at the site of only one of        the gRNA molecules. Candidate pair members can be tested for how        efficiently they remove the entire sequence versus causing indel        mutations at the target site of one gRNA molecule.

Other gRNA Design Strategy

In certain embodiments, two or more (e.g., three or four) gRNA moleculesare used with one Cas9 molecule. In another embodiment, when two or more(e.g., three or four) gRNAs are used with two or more Cas9 molecules, atleast one Cas9 molecule is from a different species than the other Cas9molecule(s). For example, when two gRNA molecules are used with two Cas9molecules, one Cas9 molecule can be from one species and the other Cas9molecule can be from a different species. Both Cas9 species are used togenerate a single or double-strand break, as desired.

In certain embodiments, dual targeting is used to create two nicks onopposite DNA strands by using Cas9 nickases (e.g., a S. pyogenes Cas9nickase) with two targeting domains that are complementary to oppositeDNA strands, e.g., a gRNA molecule comprising any minus strand targetingdomain may be paired any gRNA molecule comprising a plus strandtargeting domain provided that the two gRNAs are oriented on the DNAsuch that PAMs face outward and the distance between the 5′ ends of thegRNAs is 0-50 bp. When selecting gRNA molecules for use in a nickasepair, one gRNA molecule targets a domain in the complementary strand andthe second gRNA molecule targets a domain in the non-complementarystrand, e.g., a gRNA comprising any minus strand targeting domain may bepaired any gRNA molecule comprising a plus strand targeting domaintargeting the same target position. In certain embodiments, two 20-mergRNAs are used to target two Cas9 nucleases (e.g., two S. pyogenes Cas9nucleases) or two Cas9 nickases (e.g., two S. pyogenes Cas9 nickases).Any of the targeting domains described herein can be used with a Cas9molecule that generates a single-strand break (i.e., a S. pyogenes or S.aureus Cas9 nickase) or with a Cas9 molecule that generates adouble-strand break (i.e., S. pyogenes or S. aureus Cas9 nuclease).

gRNA molecules, as described herein, may comprise from 5′ to 3′: atargeting domain (comprising a “core domain”, and optionally a“secondary domain”); a first complementarity domain; a linking domain; asecond complementarity domain; a proximal domain; and a tail domain. Inone embodiment, the proximal domain and tail domain are taken togetheras a single domain.

In one embodiment, a gRNA molecule comprises a linking domain of no morethan 25 nucleotides in length; a proximal and tail domain, that takentogether, are at least 20 nucleotides in length; and a targeting domainequal to or greater than 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26nucleotides in length.

In another embodiment, a gRNA molecule comprises a linking domain of nomore than 25 nucleotides in length; a proximal and tail domain, thattaken together, are at least 25 nucleotides in length; and a targetingdomain equal to or greater than 16, 17, 18, 19, 20, 21, 22, 23, 24, 25or 26 nucleotides in length.

In another embodiment, a gRNA molecule comprises a linking domain of nomore than 25 nucleotides in length; a proximal and tail domain, thattaken together, are at least 30 nucleotides in length; and a targetingdomain equal to or greater than 16, 17, 18, 19, 20, 21, 22, 23, 24, 25or 26 nucleotides in length.

In another embodiment, a gRNA molecule comprises a linking domain of nomore than 25 nucleotides in length; a proximal and tail domain, thattaken together, are at least 40 nucleotides in length; and a targetingdomain equal to or greater than 16, 17, 18, 19, 20, 21, 22, 23, 24, 25or 26 nucleotides in length.

When two gRNAs are designed for use with two Cas9 molecules, the twoCas9 molecules may be from different species. Both Cas9 species may beused to generate a single or double strand break, as desired.

It is contemplated herein that any upstream gRNA described herein may bepaired with any downstream gRNA described herein. When an upstream gRNAdesigned for use with one species of Cas9 molecule is paired with adownstream gRNA designed for use from a different species of Cas9molecule, both Cas9 species are used to generate a single ordouble-strand break, as desired.

Cas9 Molecules Cas9 molecules of a variety of species can be used in themethods and compositions described herein. While S. pyogenes and S.aureus Cas9 molecules are the subject of much of the disclosure herein,Cas9 molecules of, derived from, or based on the Cas9 proteins of otherspecies listed herein can be used as well. These include, for example,Cas9 molecules from Acidovorax avenae, Actinobacillus pleuropneumonias,Actinobacillus succinogenes, Actinobacillus suis, Actinomyces sp.,cycliphilus denitrificans, Aminomonas paucivorans, Bacillus cereus,Bacillus smithii, Bacillus thuringiensis, Bacteroides sp.,Blastopirellula marina, Bradyrhizobium sp., Brevibacillus laterosporus,Campylobacter coli, Campylobacter jejuni, Campylobacter lari, CandidatusPuniceispirillum, Clostridium cellulolyticum, Clostridium perfringens,Corynebacterium accolens, Corynebacterium diphtheria, Corynebacteriummatruchotii, Dinoroseobacter shibae, Eubacterium dolichum, gammaproteobacterium, Gluconacetobacter diazotrophicus, Haemophilusparainfluenzae, Haemophilus sputorum, Helicobacter canadensis,Helicobacter cinaedi, Helicobacter mustelae, Ilyobacter polytropus,Kingella kingae, Lactobacillus crispatus, Listeria ivanovii, Listeriamonocytogenes, Listeriaceae bacterium, Methylocystis sp., Methylosinustrichosporium, Mobiluncus mulieris, Neisseria bacilliformis, Neisseriacinerea, Neisseria flavescens, Neisseria lactamica, Neisseriameningitidis, Neisseria sp., Neisseria wadsworthii, Nitrosomonas sp.,Parvibaculum lavamentivorans, Pasteurella multocida,Phascolarctobacterium succinatutens, Ralstonia syzygii, Rhodopseudomonaspalustris, Rhodovulum sp., Simonsiella muelleri, Sphingomonas sp.,Sporolactobacillus vineae, Staphylococcus lugdunensis, Streptococcussp., Subdoligranulum sp., Tistrella mobilis, Treponema sp., orVerminephrobacter eiseniae. In some embodiments, the Cas9 molecule is asplit Cas9 molecule or an inducible Cas9 molecule, as described in moredetail in WO15/089427 and WO14/018423, the entire contents of each ofwhich are expressly incorporated herein by reference.

Cas9 Domains

Crystal structures have been determined for two different naturallyoccurring bacterial Cas9 molecules (Jinek et al. 2014) and for S.pyogenes Cas9 with a guide RNA (e.g., a synthetic fusion of crRNA andtracrRNA) (Nishimasu et al. 2014; and Anders 2014).

A naturally-occurring Cas9 molecule comprises two lobes: a recognition(REC) lobe and a nuclease (NUC) lobe; each of which further comprisedomains described herein. The domain nomenclature and the numbering ofthe amino acid residues encompassed by each domain used throughout thisdisclosure is as described previously in (Nishimasu 2014). The numberingof the amino acid residues is with reference to Cas9 from S. pyogenes.

The REC lobe comprises the arginine-rich bridge helix (BH), the REC1domain, and the REC2 domain. The REC lobe does not share structuralsimilarity with other known proteins, indicating that it is aCas9-specific functional domain. The BH domain is a long a helix andarginine rich region and comprises amino acids 60-93 of the sequence ofS. pyogenes Cas9. The REC1 domain is important for recognition of therepeat:anti-repeat duplex, e.g., of a gRNA or a tracrRNA, and istherefore critical for Cas9 activity by recognizing the target sequence.The REC1 domain comprises two REC1 motifs at amino acids 94 to 179 and308 to 717 of the sequence of S. pyogenes Cas9. These two REC1 domains,though separated by the REC2 domain in the linear primary structure,assemble in the tertiary structure to form the REC1 domain. The REC2domain, or parts thereof, may also play a role in the recognition of therepeat:anti-repeat duplex. The REC2 domain comprises amino acids 180-307of the sequence of S. pyogenes Cas9.

The NUC lobe comprises the RuvC domain, the HNH domain, and thePAM-interacting (PI) domain. The RuvC domain shares structuralsimilarity to retroviral integrase superfamily members and cleaves asingle strand, e.g., the non-complementary strand of the target nucleicacid molecule. The RuvC domain is assembled from the three split RuvCmotifs (RuvC I, RuvCII, and RuvCIII, which are often commonly referredto in the art as RuvCI domain, or N-terminal RuvC domain, RuvCII domain,and RuvCIII domain) at amino acids 1-59, 718-769, and 909-1098,respectively, of the sequence of S. pyogenes Cas9. Similar to the REC1domain, the three RuvC motifs are linearly separated by other domains inthe primary structure, however in the tertiary structure, the three RuvCmotifs assemble and form the RuvC domain. The HNH domain sharesstructural similarity with HNH endonucleases, and cleaves a singlestrand, e.g., the complementary strand of the target nucleic acidmolecule. The HNH domain lies between the RuvC II-III motifs andcomprises amino acids 775-908 of the sequence of S. pyogenes Cas9. ThePI domain interacts with the PAM of the target nucleic acid molecule,and comprises amino acids 1099-1368 of the sequence of S. pyogenes Cas9.

RuvC-Like Domain and an HNH-Like Domain

In certain embodiments, a Cas9 molecule or Cas9 polypeptide comprises anHNH-like domain and a RuvC-like domain and in certain of theseembodiments cleavage activity is dependent on the RuvC-like domain andthe HNH-like domain. A Cas9 molecule or Cas9 polypeptide can compriseone or more of a RuvC-like domain and an HNH-like domain. In certainembodiments, a Cas9 molecule or Cas9 polypeptide comprises a RuvC-likedomain, e.g., a RuvC-like domain described below, and/or an HNH-likedomain, e.g., an HNH-like domain described below.

RuvC-Like Domains

In certain embodiments, a RuvC-like domain cleaves, a single strand,e.g., the non-complementary strand of the target nucleic acid molecule.The Cas9 molecule or Cas9 polypeptide can include more than oneRuvC-like domain (e.g., one, two, three or more RuvC-like domains). Incertain embodiments, a RuvC-like domain is at least 5, 6, 7, 8 aminoacids in length but not more than 20, 19, 18, 17, 16 or 15 amino acidsin length. In certain embodiments, the Cas9 molecule or Cas9 polypeptidecomprises an N-terminal RuvC-like domain of about 10 to 20 amino acids,e.g., about 15 amino acids in length.

N-Terminal RuvC-Like Domains

Some naturally occurring Cas9 molecules comprise more than one RuvC-likedomain with cleavage being dependent on the N-terminal RuvC-like domain.Accordingly, a Cas9 molecule or Cas9 polypeptide can comprise anN-terminal RuvC-like domain. Exemplary N-terminal RuvC-like domains aredescribed below.

In certain embodiments, a Cas9 molecule or Cas9 polypeptide comprises anN-terminal RuvC-like domain comprising an amino acid sequence of FormulaI:

(SEQ ID NO: 8) D-X₁-G-X₂-X₃-X₄-X₅-G-X₆-X₇-X₈-X₉,wherein,

X₁ is selected from I, V, M, L and T (e.g., selected from I, V, and L);

X₂ is selected from T, I, V, S, N, Y, E and L (e.g., selected from T, V,and I);

X₃ is selected from N, S, G, A, D, T, R, M and F (e.g., A or N);

X₄ is selected from S, Y, N and F (e.g., S);

X₅ is selected from V, I, L, C, T and F (e.g., selected from V, I andL);

X₆ is selected from W, F, V, Y, S and L (e.g., W);

X₇ is selected from A, S, C, V and G (e.g., selected from A and S);

X₈ is selected from V, I, L, A, M and H (e.g., selected from V, I, M andL); and

X₉ is selected from any amino acid or is absent (e.g., selected from T,V, I, L, A, F, S, A, Y, M and R, or, e.g., selected from T, V, I, L andA).

In certain embodiments, the N-terminal RuvC-like domain differs from asequence of SEQ ID NO:8, by as many as 1 but no more than 2, 3, 4, or 5residues.

In certain embodiments, the N-terminal RuvC-like domain is cleavagecompetent.

In other embodiments, the N-terminal RuvC-like domain is cleavageincompetent.

In certain embodiments, a Cas9 molecule or Cas9 polypeptide comprises anN-terminal RuvC-like domain comprising an amino acid sequence of FormulaII:

(SEQ ID NO: 9) D-X₁-G-X₂-X₃-S-X₅-G-X₆-X₇-X₈-X₉,,wherein

X₁ is selected from I, V, M, L and T (e.g., selected from I, V, and L);

X₂ is selected from T, I, V, S, N, Y, E and L (e.g., selected from T, V,and I);

X₃ is selected from N, S, G, A, D, T, R, M and F (e.g., A or N);

X₅ is selected from V, I, L, C, T and F (e.g., selected from V, I andL);

X₆ is selected from W, F, V, Y, S and L (e.g., W);

X₇ is selected from A, S, C, V and G (e.g., selected from A and 5);

X₈ is selected from V, I, L, A, M and H (e.g., selected from V, I, M andL); and

X₉ is selected from any amino acid or is absent (e.g., selected from T,V, I, L, A, F, S, A, Y, M and R or selected from e.g., T, V, I, L andA).

In certain embodiments, the N-terminal RuvC-like domain differs from asequence of SEQ ID NO:9 by as many as 1 but not more than 2, 3, 4, or 5residues.

In certain embodiments, the N-terminal RuvC-like domain comprises anamino acid sequence of Formula III:

(SEQ ID NO: 10) D-I-G-X₂-X₃-S-V-G-W-A-X₈-X₉,wherein

X₂ is selected from T, I, V, S, N, Y, E and L (e.g., selected from T, V,and I);

X₃ is selected from N, S, G, A, D, T, R, M and F (e.g., A or N);

X₈ is selected from V, I, L, A, M and H (e.g., selected from V, I, M andL); and

X₉ is selected from any amino acid or is absent (e.g., selected from T,V, I, L, A, F, S, A, Y, M and R or selected from e.g., T, V, I, L andA).

In certain embodiments, the N-terminal RuvC-like domain differs from asequence of SEQ ID NO:10 by as many as 1 but not more than, 2, 3, 4, or5 residues.

In certain embodiments, the N-terminal RuvC-like domain comprises anamino acid sequence of Formula IV:

(SEQ ID NO: 11) D-I-G-T-N-S-V-G-W-A-V-X,wherein

X is a non-polar alkyl amino acid or a hydroxyl amino acid, e.g., X isselected from V, I, L and T.

In certain embodiments, the N-terminal RuvC-like domain differs from asequence of SEQ ID NO:11 by as many as 1 but not more than, 2, 3, 4, or5 residues.

In certain embodiments, the N-terminal RuvC-like domain differs from asequence of an N-terminal RuvC like domain disclosed herein, e.g., inany one of SEQ ID Nos: 54-103, as many as 1 but no more than 2, 3, 4, or5 residues. In certain embodiments, 1, 2, 3 or all of the highlyconserved residues of SEQ ID Nos: 54-103 are present.

In certain embodiment, the N-terminal RuvC-like domain differs from asequence of an N-terminal RuvC-like domain disclosed herein, e.g., inany one of SEQ ID Nos: 104-177, as many as 1 but no more than 2, 3, 4,or 5 residues. In certain embodiments, 1, 2, or all of the highlyconserved residues identified of SEQ ID Nos: 104-177 are present.

Additional RuvC-Like Domains

In addition to the N-terminal RuvC-like domain, the Cas9 molecule orCas9 polypeptide can comprise one or more additional RuvC-like domains.In certain embodiments, the Cas9 molecule or Cas9 polypeptide cancomprise two additional RuvC-like domains. Preferably, the additionalRuvC-like domain is at least 5 amino acids in length and, e.g., lessthan 15 amino acids in length, e.g., 5 to 10 amino acids in length,e.g., 8 amino acids in length.

An additional RuvC-like domain can comprise an amino acid sequence ofFormula V:

(SEQ ID NO: 12) I-X₁-X₂-E-X₃-A-R-E,

wherein

X₁ is V or H;

X₂ is I, L or V (e.g., I or V); and

X₃ is M or T.

In certain embodiments, the additional RuvC-like domain comprises anamino acid sequence of Formula VI:

(SEQ ID NO: 13) I-V-X₂-E-M-A-R-E,wherein

X₂ is L or V (e.g., I or V).

An additional RuvC-like domain can comprise an amino acid sequence ofFormula VII:

(SEQ ID NO: 14) H-H-A-X₁-D-A-X₂-X₃,wherein

X₁ is H or L;

X₂ is R or V; and

X₃ is E or V.

In certain embodiments, the additional RuvC-like domain comprises theamino acid sequence: H-H-A-H-D-A-Y-L (SEQ ID NO:15).

In certain embodiments, the additional RuvC-like domain differs from asequence of SEQ ID NOs: 12-15 by as many as 1 but not more than 2, 3, 4,or 5 residues.

In certain embodiment, the sequence flanking the N-terminal RuvC-likedomain has the amino acid sequence of Formula VIII:

(SEQ ID NO: 16) K-X₁-Y-X₂′-X₃′-X₄′-Z-T-D-X₉′-Y,.wherein

X₁′ is selected from K and P;

X₂′ is selected from V, L, I, and F (e.g., V, I and L);

X₃′ is selected from G, A and S (e.g., G);

X₄′ is selected from L, I, V and F (e.g., L);

X₉′ is selected from D, E, N and Q; and

Z is an N-terminal RuvC-like domain, e.g., as described above, e.g.,having 5 to 20 amino acids.

HNH-Like Domains

In certain embodiments, an HNH-like domain cleaves a single strandedcomplementary domain, e.g., a complementary strand of a double strandednucleic acid molecule. In certain embodiments, an HNH-like domain is atleast 15, 20, or 25 amino acids in length but not more than 40, 35, or30 amino acids in length, e.g., 20 to 35 amino acids in length, e.g., 25to 30 amino acids in length. Exemplary HNH-like domains are describedbelow.

In certain embodiments, a Cas9 molecule or Cas9 polypeptide comprises anHNH-like domain having an amino acid sequence of Formula IX:

(SEQ ID NO: 17) X₁-X₂-X₃-H-X₄-X₅-P-X₆-X₇-X₈-X₉-X₁₀-X₁₁-X₁₂-X₁₃-X₁₄-X₁₅-N-X₁₆-X₁₇-X₁₈-X₁₉-X₂₀-X₂₁-X₂₂-X₂₃-N, wherein

X₁ is selected from D, E, Q and N (e.g., D and E);

X₂ is selected from L, I, R, Q, V, M and K;

X₃ is selected from D and E;

X₄ is selected from I, V, T, A and L (e.g., A, I and V);

X₅ is selected from V, Y, I, L, F and W (e.g., V, I and L);

X₆ is selected from Q, H, R, K, Y, I, L, F and W;

X₇ is selected from S, A, D, T and K (e.g., S and A);

X₈ is selected from F, L, V, K, Y, M, I, R, A, E, D and Q (e.g., F);

X₉ is selected from L, R, T, I, V, S, C, Y, K, F and G;

X₁₀ is selected from K, Q, Y, T, F, L, W, M, A, E, G, and S;

X₁₁ is selected from D, S, N, R, L and T (e.g., D);

X₁₂ is selected from D, N and S;

X₁₃ is selected from S, A, T, G and R (e.g., S);

X₁₄ is selected from I, L, F, S, R, Y, Q, W, D, K and H (e.g., I, L andF);

X₁₅ is selected from D, S, I, N, E, A, H, F, L, Q, M, G, Y and V;

X₁₆ is selected from K, L, R, M, T and F (e.g., L, R and K);

X₁₇ is selected from V, L, I, A and T;

X₁₈ is selected from L, I, V and A (e.g., L and I);

X₁₉ is selected from T, V, C, E, S and A (e.g., T and V);

X₂₀ is selected from R, F, T, W, E, L, N, C, K, V, S, Q, I, Y, H and A;

X₂₁ is selected from S, P, R, K, N, A, H, Q, G and L;

X₂₂ is selected from D, G, T, N, S, K, A, I, E, L, Q, R and Y; and

X₂₃ is selected from K, V, A, E, Y, I, C, L, S, T, G, K, M, D and F.

In certain embodiments, a HNH-like domain differs from a sequence of SEQID NO: 17 by at least one but not more than, 2, 3, 4, or 5 residues.

In certain embodiments, the HNH-like domain is cleavage competent.

In other embodiments, the HNH-like domain is cleavage incompetent.

In certain embodiments, a Cas9 molecule or Cas9 polypeptide comprises anHNH-like domain comprising an amino acid sequence of Formula X:

(SEQ ID NO: 18) X₁-X₂-X₃-H-X₄-X₅-P-X₆-S-X₈-X₉-X₁₀-D-D-S-X₁₄-X₁₅-N-K-V-L-X₁₉-X₂₀-X₂₁-X₂₂-X₂₃-N, wherein

X₁ is selected from D and E;

X₂ is selected from L, I, R, Q, V, M and K;

X₃ is selected from D and E;

X₄ is selected from I, V, T, A and L (e.g., A, I and V);

X₅ is selected from V, Y, I, L, F and W (e.g., V, I and L);

X₆ is selected from Q, H, R, K, Y, I, L, F and W;

X₈ is selected from F, L, V, K, Y, M, I, R, A, E, D and Q (e.g., F);

X₉ is selected from L, R, T, I, V, S, C, Y, K, F and G;

X₁₀ is selected from K, Q, Y, T, F, L, W, M, A, E, G, and S;

X₁₄ is selected from I, L, F, S, R, Y, Q, W, D, K and H (e.g., I, L andF);

X₁₅ is selected from D, S, I, N, E, A, H, F, L, Q, M, G, Y and V;

X₁₉ is selected from T, V, C, E, S and A (e.g., T and V);

X₂₀ is selected from R, F, T, W, E, L, N, C, K, V, S, Q, I, Y, H and A;

X₂₁ is selected from S, P, R, K, N, A, H, Q, G and L;

X₂₂ is selected from D, G, T, N, S, K, A, I, E, L, Q, R and Y; and

X₂₃ is selected from K, V, A, E, Y, I, C, L, S, T, G, K, M, D and F.

In certain embodiments, the HNH-like domain differs from a sequence ofSEQ ID NO: 18 by 1, 2, 3, 4, or 5 residues.

In certain embodiments, a Cas9 molecule or Cas9 polypeptide comprises anHNH-like domain comprising an amino acid sequence of Formula XI:

(SEQ ID NO: 19) X₁-V-X₃-H-I-V-P-X₆-S-X₈-X₉-X₁₀-D-D-S-X₁₄-X₁₅-N-K-V-L-T-X₂₀-X₂₁-X₂₂-X₂₃-N,wherein

X₁ is selected from D and E;

X₃ is selected from D and E;

X₆ is selected from Q, H, R, K, Y, I, L and W;

X₈ is selected from F, L, V, K, Y, M, I, R, A, E, D and Q (e.g., F);

X₉ is selected from L, R, T, I, V, S, C, Y, K, F and G;

X₁₀ is selected from K, Q, Y, T, F, L, W, M, A, E, G, and S;

X₁₄ is selected from I, L, F, S, R, Y, Q, W, D, K and H (e.g., I, L andF);

X₁₅ is selected from D, S, I, N, E, A, H, F, L, Q, M, G, Y and V;

X₂₀ is selected from R, F, T, W, E, L, N, C, K, V, S, Q, I, Y, H and A;

X₂₁ is selected from S, P, R, K, N, A, H, Q, G and L;

X₂₂ is selected from D, G, T, N, S, K, A, I, E, L, Q, R and Y; and

X₂₃ is selected from K, V, A, E, Y, I, C, L, S, T, G, K, M, D and F.

In certain embodiments, the HNH-like domain differs from a sequence ofSEQ ID NO: 19 by 1, 2, 3, 4, or 5 residues.

In certain embodiments, a Cas9 molecule or Cas9 polypeptide comprises anHNH-like domain having an amino acid sequence of Formula XII:

(SEQ ID NO: 20) D-X₂-D-H-I-X₅-P-Q-X₇-F-X₉-X₁₀-D-X₁₂-S-I-D-N-X₁₆-V-L-X₁₉-X₂₀-S-X₂₂-X₂₃-N,wherein

X₂ is selected from I and V;

X₅ is selected from I and V;

X₇ is selected from A and S;

X₉ is selected from I and L;

X₁₀ is selected from K and T;

X₁₂ is selected from D and N;

X₁₆ is selected from R, K and L;

X₁₉ is selected from T and V;

X₂₀ is selected from S and R;

X₂₂ is selected from K, D and A; and

X₂₃ is selected from E, K, G and N (e.g., the Cas9 molecule or Cas9polypeptide can comprise an HNH-like domain as described herein).

In certain embodiments, the HNH-like domain differs from a sequence ofSEQ ID NO: 20 by as many as 1 but not more than 2, 3, 4, or 5 residues.

In certain embodiments, a Cas9 molecule or Cas9 polypeptide comprisesthe amino acid sequence of formula XIII:

(SEQ ID NO: 21) L-Y-Y-L-Q-N-G-X₁′-D-M-Y-X₂′-X₃′-X₄′-X₅′-L-D-I-X₆′-X₇′-L-S-X₈′-Y-Z-N-R-X₉′-K-X₁₀′-D-X₁₁′-V-P, wherein

X₁′ is selected from K and R;

X₂′ is selected from V and T;

X₃′ is selected from G and D;

X₄′ is selected from E, Q and D;

X₅′ is selected from E and D;

X₆′ is selected from D, N and H;

X₇′ is selected from Y, R and N;

X₈′ is selected from Q, D and N;

X₉′ is selected from G and E;

X₁₀′ is selected from S and G;

X₁₁′ is selected from D and N; and

Z is an HNH-like domain, e.g., as described above.

In certain embodiment, a Cas9 molecule or Cas9 polypeptide comprises anamino acid sequence that differs from a sequence of SEQ ID NO:21 by asmany as 1 but not more than 2, 3, 4, or 5 residues.

In certain embodiments, the HNH-like domain differs from a sequence ofan HNH-like domain disclosed herein by as many as 1 but not more than 2,3, 4, or 5 residues. In certain embodiments, 1 or both of the highlyconserved residues of are present.

In certain embodiments, the HNH-like domain differs from a sequence ofan HNH-like domain disclosed herein by as many as 1 but not more than 2,3, 4, or 5 residues. In certain embodiments, 1, 2, all 3 of the highlyconserved residues are present.

Inducible Cas9 Molecules and Gene Editing Systems

In some embodiments, the Cas9 fusion molecule comprises an inducibleCas9 molecule, as described in more detail in WO15/089427 andWO14/018423, the entire contents of each of which are expresslyincorporated herein by reference. Inducible Cas9 molecules aresummarized briefly, below.

In one aspect, disclosed herein is a non-naturally occurring orengineered gene editing system, comprising a Cas9 molecule, which maycomprise at least one switch, wherein the activity of said gene editingsystem is controlled by contact with at least one inducer energy sourceas to the switch. In an embodiment, the control as to the at least oneswitch or the activity of the gene editing system may be activated,enhanced, terminated or repressed. The contact with the at least oneinducer energy source may result in a first effect and a second effect.The first effect may be one or more of nuclear import, nuclear export,recruitment of a secondary component (such as an effector molecule),conformational change (of protein, DNA or RNA), cleavage, release ofcargo (such as a caged molecule or a co-factor), association ordissociation. The second effect may be one or more of activation,enhancement, termination or repression of the control as to the at leastone switch or the activity of the gene editing system. In oneembodiment, the first effect and the second effect may occur in acascade.

In one embodiment, the Cas9 molecule may further comprise at least onenuclear localization signal (NLS), nuclear export signal (NES),functional domain, flexible linker, mutation, deletion, alteration ortruncation. The one or more of the NLS, the NES or the functional domainmay be conditionally activated or inactivated. In another embodiment,the mutation may be one or more of a mutation in a transcription factorhomology region, a mutation in a DNA binding domain (such as mutatingbasic residues of a basic helix loop helix), a mutation in an endogenousNLS or a mutation in an endogenous NES. The disclosure comprehends thatthe inducer energy source may be heat, ultrasound, electromagneticenergy or chemical. In a preferred embodiment of the invention, theinducer energy source may be an antibiotic, a small molecule, a hormone,a hormone derivative, a steroid or a steroid derivative. In a morepreferred embodiment, the inducer energy source maybe abscisic acid(ABA), doxycycline (DOX), cumate, rapamycin, 4-hydroxytamoxifen (40HT),estrogen or ecdysone. The disclosure also provides that the at least oneswitch may be selected from the group consisting of antibiotic basedinducible systems, electromagnetic energy based inducible systems, smallmolecule based inducible systems, nuclear receptor based induciblesystems and hormone based inducible systems. In a more preferredembodiment, the at least one switch may be selected from the groupconsisting of tetracycline (Tet)/DOX inducible systems, light induciblesystems, ABA inducible systems, cumate repressor/operator systems,40HT/estrogen inducible systems, ecdysone-based inducible systems andFKBP12/FRAP (FKBP12-rapamycin complex) inducible systems.

The at least one functional domain may be selected from the groupconsisting of: transposase domain, integrase domain, recombinase domain,resolvase domain, invertase domain, protease domain, DNAmethyltransferase domain, DNA hydroxylmethylase domain, DNA demethylasedomain, histone acetylase domain, histone deacetylases domain, nucleasedomain, repressor domain, activator domain, nuclear-localization signaldomains, transcription-regulatory protein (or transcription complexrecruiting) domain, cellular uptake activity associated domain, nucleicacid binding domain, antibody presentation domain, histone modifyingenzymes, recruiter of histone modifying enzymes; inhibitor of histonemodifying enzymes, histone methyltransferase, histone demethylase,histone kinase, histone phosphatase, histone ribosylase, histonederibosylase, histone ubiquitinase, histone deubiquitinase, histonebiotinase or histone tail protease.

Specifically, the disclosure provides for systems or methods asdescribed herein, wherein the gene editing system may comprise a vectorsystem comprising: a) a first regulatory element operably linked to agene editing system guide RNA that targets a locus of interest, b) asecond regulatory inducible element operably linked to a Cas9 fusionprotein, wherein components (a) and (b) may be located on same ordifferent vectors of the system, wherein the guide RNA targets DNA ofthe locus of interest, wherein the Cas9 fusion protein and the guide RNAdo not naturally occur together. In a preferred embodiment of theinvention, the Cas9 fusion protein comprises an inducible Cas9 enzyme.The invention also provides for the vector being a AAV or a lentivirus.

Split Cas9 Molecules and Gene Editing Systems

In some embodiments, the Cas9 fusion molecule comprises a split Cas9molecule, as described in more detail in WO15/089427 and WO14/018423,the entire contents of each of which are expressly incorporated hereinby reference. Split Cas9 molecules are summarized briefly, below.

In an aspect, disclosed herein is a non-naturally occurring orengineered inducible CRISPR enzyme, e.g., Cas9 enzyme, comprising: afirst CRISPR enzyme fusion construct attached to a first half of aninducible dimer and a second CRISPR enzyme fusion construct attached toa second half of the inducible dimer, wherein the first CRISPR enzymefusion construct is operably linked to one or more nuclear localizationsignals, wherein the second CRISPR enzyme fusion construct is operablylinked to one or more nuclear export signals, wherein contact with aninducer energy source brings the first and second halves of theinducible dimer together, wherein bringing the first and second halvesof the inducible dimer together allows the first and second CRISPRenzyme fusion constructs to constitute a functional gene editing system.

In another aspect, in the inducible gene editing system, the inducibledimer is or comprises or consists essentially of or consists of aninducible heterodimer. In an aspect, in inducible gene editing system,the first half or a first portion or a first fragment of the inducibleheterodimer is or comprises or consists of or consists essentially of anFKBP, optionally FKBP 12. In an aspect, in the inducible gene editingsystem, the second half or a second portion or a second fragment of theinducible heterodimer is or comprises or consists of or consistsessentially of FRB. In one aspect, in the inducible gene editing system,the arrangement of the first CRISPR enzyme fusion construct is orcomprises or consists of or consists essentially of N′ terminal Cas9part-FRB-NES. In another aspect, in the inducible gene editing system,the arrangement of the first CRISPR enzyme fusion construct is orcomprises or consists of or consists essentially of NES-N′ terminal Cas9part-FRB-NES. In one aspect in the inducible gene editing system, thearrangement of the second CRISPR enzyme fusion construct is or comprisesor consists essentially of or consists of C terminal Cas9 part-FKBP-NLS.In another aspect, in the inducible gene editing system, the arrangementof the second CRISPR enzyme fusion construct is or comprises or consistsof or consists essentially of NLS-C terminal Cas9 part-FKBP-NLS. In anaspect, in inducible gene editing system there can be a linker thatseparates the Cas9 part from the half or portion or fragment of theinducible dimer. In an aspect, in the inducible gene editing system, theinducer energy source is or comprises or consists essentially of orconsists of rapamycin. In an aspect, in inducible gene editing system,the inducible dimer is an inducible homodimer. In an aspect, ininducible gene editing system, the CRISPR enzyme is Cas9, e.g., SpCas9or SaCas9. In an aspect in an gene editing system, the Cas9 is splitinto two parts at any one of the following split points, according orwith reference to SpCas9: a split position between 202A/203S; a splitposition between 255F/256D; a split position between 310E/311I; a splitposition between 534R/535; a split position between 572E/573C; a splitposition between 713S/714G; a split position between 1003L/104E; a splitposition between 1 G54G/1 Q55E; a split position between 11 14N/1115S; asplit position between 1152K/1153S; a split position between1245K/1246G; or a split between 1098 and 1099. In an aspect, in theinducible gene editing system, one or more functional domains areassociated with one or both parts of the Cas9 enzyme, e.g., thefunctional domains optionally including a transcriptional activator, atranscriptional or a nuclease such as a fok I nuclease. In an aspect, inthe inducible gene editing system, the functional gene editing systembinds to the target sequence and the enzyme is a deadCas9, optionallyhaving a diminished nuclease activity of at least 97%, or 100% (or nomore than 3% and advantageously 0%) nuclease activity) as compared withthe CRISPR enzyme not having the at least one mutation. In an aspect, inthe inducible gene editing system, the deadCas9 (CRISPR enzyme)comprises two or more mutations wherein two or more of DIG, E762, H840,N854, N863, or D986 according to SpCas9 protein or any correspondingortholog or N580 according to SaCas9 protein are mutated, or the CRISPRenzyme comprises at least one mutation, e.g., wherein at least H840 ismutated. The disclosure further provides, a polynucleotide encoding theinducible gene editing system as herein discussed.

Also disclosed herein is a vector for delivery of the first CRISPRenzyme fusion construct, attached to a first half or portion or fragmentof an inducible dimer and operably linked to one or more nuclearlocalization signals, according as herein discussed. In an aspect,disclosed herein is a vector for delivery of the second CRISPR enzymefusion construct, attached to a second half or portion or fragment of aninducible dimer and operably linked to one or more nuclear exportsignals.

Cas9 Activities

In certain embodiments, the Cas9 molecule or Cas9 polypeptide is capableof cleaving a target nucleic acid molecule. Typically wild-type Cas9molecules cleave both strands of a target nucleic acid molecule. Cas9molecules and Cas9 polypeptides can be engineered to alter nucleasecleavage (or other properties), e.g., to provide a Cas9 molecule or Cas9polypeptide which is a nickase, or which lacks the ability to cleavetarget nucleic acid. A Cas9 molecule or Cas9 polypeptide that is capableof cleaving a target nucleic acid molecule is referred to herein as aneaCas9 (an enzymatically active Cas9) molecule or eaCas9 polypeptide.

In certain embodiments, an eaCas9 molecule or eaCas9 polypeptidecomprises one or more of the following enzymatic activities:

a nickase activity, i.e., the ability to cleave a single strand, e.g.,the non-complementary strand or the complementary strand, of a nucleicacid molecule;

a double stranded nuclease activity, i.e., the ability to cleave bothstrands of a double stranded nucleic acid and create a double strandedbreak, which in one embodiment is the presence of two nickaseactivities;

an endonuclease activity;

an exonuclease activity; and

a helicase activity, i.e., the ability to unwind the helical structureof a double stranded nucleic acid.

In certain embodiments, an enzymatically active Cas9 or eaCas9 moleculeor eaCas9 polypeptide cleaves both DNA strands and results in a doublestranded break. In certain embodiments, an eaCas9 molecule or eaCas9polypeptide cleaves only one strand, e.g., the strand to which the gRNAhybridizes to, or the strand complementary to the strand the gRNAhybridizes with. In one embodiment, an eaCas9 molecule or eaCas9polypeptide comprises cleavage activity associated with an HNH domain.In one embodiment, an eaCas9 molecule or eaCas9 polypeptide comprisescleavage activity associated with a RuvC domain. In one embodiment, aneaCas9 molecule or eaCas9 polypeptide comprises cleavage activityassociated with an HNH domain and cleavage activity associated with aRuvC domain. In one embodiment, an eaCas9 molecule or eaCas9 polypeptidecomprises an active, or cleavage competent, HNH domain and an inactive,or cleavage incompetent, RuvC domain. In one embodiment, an eaCas9molecule or eaCas9 polypeptide comprises an inactive, or cleavageincompetent, HNH domain and an active, or cleavage competent, RuvCdomain.

Some Cas9 molecules or Cas9 polypeptides have the ability to interactwith a gRNA molecule, and in conjunction with the gRNA molecule localizeto a core target domain, but are incapable of cleaving the targetnucleic acid, or incapable of cleaving at efficient rates. Cas9molecules having no, or no substantial, cleavage activity are referredto herein as an eiCas9 molecule or eiCas9 polypeptide. For example, aneiCas9 molecule or eiCas9 polypeptide can lack cleavage activity or havesubstantially less, e.g., less than 20, 10, 5, 1 or 0.1% of the cleavageactivity of a reference Cas9 molecule or eiCas9 polypeptide, as measuredby an assay described herein.

Targeting and PAMs

A Cas9 molecule or Cas9 polypeptide that can interact with a gRNAmolecule and, in concert with the gRNA molecule, localizes to a sitewhich comprises a target domain, and in certain embodiments, a PAMsequence.

In certain embodiments, the ability of an eaCas9 molecule or eaCas9polypeptide to interact with and cleave a target nucleic acid is PAMsequence dependent. A PAM sequence is a sequence in the target nucleicacid. In one embodiment, cleavage of the target nucleic acid occursupstream from the PAM sequence. eaCas9 molecules from differentbacterial species can recognize different sequence motifs (e.g., PAMsequences). In one embodiment, an eaCas9 molecule of S. pyogenesrecognizes the sequence motif NGG and directs cleavage of a targetnucleic acid sequence 1 to 10, e.g., 3 to 5, bp upstream from thatsequence (see, e.g., Mali 2013). In one embodiment, an eaCas9 moleculeof S. thermophilus recognizes the sequence motif NGGNG and/or NNAGAAW(W=A or T) and directs cleavage of a target nucleic acid sequence 1 to10, e.g., 3 to 5, bp upstream from these sequences (see, e.g., Horvath2010; Deveau 2008). In one embodiment, an eaCas9 molecule of S. mutansrecognizes the sequence motif NGG and/or NAAR (R=A or G) and directscleavage of a target nucleic acid sequence 1 to 10, e.g., 3 to 5, bpupstream from this sequence (see, e.g., Deveau 2008). In one embodiment,an eaCas9 molecule of S. aureus recognizes the sequence motif NNGRR (R=Aor G) and directs cleavage of a target nucleic acid sequence 1 to 10,e.g., 3 to 5, bp upstream from that sequence. In one embodiment, aneaCas9 molecule of S. aureus recognizes the sequence motif NNGRRN (R=Aor G) and directs cleavage of a target nucleic acid sequence 1 to 10,e.g., 3 to 5, bp upstream from that sequence. In one embodiment, aneaCas9 molecule of S. aureus recognizes the sequence motif NNGRRT (R=Aor G) and directs cleavage of a target nucleic acid sequence 1 to 10,e.g., 3 to 5, base pairs upstream from that sequence. In one embodiment,an eaCas9 molecule of S. aureus recognizes the sequence motif NNGRRV(R=A or G) and directs cleavage of a target nucleic acid sequence 1 to10, e.g., 3 to 5, bp upstream from that sequence. The ability of a Cas9molecule to recognize a PAM sequence can be determined, e.g., using atransformation assay as described in Jinek 2012. In the aforementionedembodiments, N can be any nucleotide residue, e.g., any of A, G, C, orT.

As is discussed herein, Cas9 molecules can be engineered to alter thePAM specificity of the Cas9 molecule.

Exemplary naturally occurring Cas9 molecules have been describedpreviously (see, e.g., Chylinski 2013). Such Cas9 molecules include Cas9molecules of a cluster 1 bacterial family, cluster 2 bacterial family,cluster 3 bacterial family, cluster 4 bacterial family, cluster 5bacterial family, cluster 6 bacterial family, a cluster 7 bacterialfamily, a cluster 8 bacterial family, a cluster 9 bacterial family, acluster 10 bacterial family, a cluster 11 bacterial family, a cluster 12bacterial family, a cluster 13 bacterial family, a cluster 14 bacterialfamily, a cluster 15 bacterial family, a cluster 16 bacterial family, acluster 17 bacterial family, a cluster 18 bacterial family, a cluster 19bacterial family, a cluster 20 bacterial family, a cluster 21 bacterialfamily, a cluster 22 bacterial family, a cluster 23 bacterial family, acluster 24 bacterial family, a cluster 25 bacterial family, a cluster 26bacterial family, a cluster 27 bacterial family, a cluster 28 bacterialfamily, a cluster 29 bacterial family, a cluster 30 bacterial family, acluster 31 bacterial family, a cluster 32 bacterial family, a cluster 33bacterial family, a cluster 34 bacterial family, a cluster 35 bacterialfamily, a cluster 36 bacterial family, a cluster 37 bacterial family, acluster 38 bacterial family, a cluster 39 bacterial family, a cluster 40bacterial family, a cluster 41 bacterial family, a cluster 42 bacterialfamily, a cluster 43 bacterial family, a cluster 44 bacterial family, acluster 45 bacterial family, a cluster 46 bacterial family, a cluster 47bacterial family, a cluster 48 bacterial family, a cluster 49 bacterialfamily, a cluster 50 bacterial family, a cluster 51 bacterial family, acluster 52 bacterial family, a cluster 53 bacterial family, a cluster 54bacterial family, a cluster 55 bacterial family, a cluster 56 bacterialfamily, a cluster 57 bacterial family, a cluster 58 bacterial family, acluster 59 bacterial family, a cluster 60 bacterial family, a cluster 61bacterial family, a cluster 62 bacterial family, a cluster 63 bacterialfamily, a cluster 64 bacterial family, a cluster 65 bacterial family, acluster 66 bacterial family, a cluster 67 bacterial family, a cluster 68bacterial family, a cluster 69 bacterial family, a cluster 70 bacterialfamily, a cluster 71 bacterial family, a cluster 72 bacterial family, acluster 73 bacterial family, a cluster 74 bacterial family, a cluster 75bacterial family, a cluster 76 bacterial family, a cluster 77 bacterialfamily, or a cluster 78 bacterial family.

Exemplary naturally occurring Cas9 molecules include a Cas9 molecule ofa cluster 1 bacterial family. Examples include a Cas9 molecule of: S.aureus, S. pyogenes (e.g., strain SF370, MGAS10270, MGAS10750, MGAS2096,MGAS315, MGAS5005, MGAS6180, MGAS9429, NZ131 and SSI-1), S. thermophilus(e.g., strain LMD-9), S. pseudoporcinus (e.g., strain SPIN 20026), S.mutans (e.g., strain UA159, NN2025), S. macacae (e.g., strainNCTC11558), S. gallolyticus (e.g., strain UCN34, ATCC BAA-2069), S.equines (e.g., strain ATCC 9812, MGCS 124), S. dysdalactiae (e.g.,strain GGS 124), S. bovis (e.g., strain ATCC 700338), S. anginosus(e.g., strain F0211), S. agalactiae (e.g., strain NEM316, A909),Listeria monocytogenes (e.g., strain F6854), Listeria innocua (L.innocua, e.g., strain Clip11262), Enterococcus italicus (e.g., strainDSM 15952), or Enterococcus faecium (e.g., strain 1,231,408).

In certain embodiments, a Cas9 molecule or Cas9 polypeptide comprises anamino acid sequence: having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,97%, 98% or 99% homology with; differs at no more than, 2, 5, 10, 15,20, 30, or 40% of the amino acid residues when compared with; differs byat least 1, 2, 5, 10 or 20 amino acids, but by no more than 100, 80, 70,60, 50, 40 or 30 amino acids from; or is identical to any Cas9 moleculesequence described herein, or to a naturally occurring Cas9 moleculesequence, e.g., a Cas9 molecule from a species listed herein (e.g., SEQID NO:1-4 or described in Chylinski 2013 or Hou 2013). In oneembodiment, the Cas9 molecule or Cas9 polypeptide comprises one or moreof the following activities: a nickase activity; a double strandedcleavage activity (e.g., an endonuclease and/or exonuclease activity); ahelicase activity; or the ability, together with a gRNA molecule, tolocalize to a target nucleic acid.

A comparison of the sequence of a number of Cas9 molecules indicate thatcertain regions are conserved. These are identified below as:

region 1 (residues 1 to 180, or in the case of region 1, residues 120 to180) region 2 (residues 360 to 480);

region 3 (residues 660 to 720);

region 4 (residues 817 to 900); and

region 5 (residues 900 to 960).

In one embodiment, a Cas9 molecule or Cas9 polypeptide comprises regions1-5, together with sufficient additional Cas9 molecule sequence toprovide a biologically active molecule, e.g., a Cas9 molecule having atleast one activity described herein. In one embodiment, each of regions1-5, independently, have 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%,98% or 99% homology with the corresponding residues of a Cas9 moleculeor Cas9 polypeptide described herein, e.g., a sequence from SEQ ID Nos:1-4.

In one embodiment, a Cas9 molecule or Cas9 polypeptide comprises anamino acid sequence referred to as region 1:

having 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homologywith amino acids 1-180 of the amino acid sequence of Cas9 of S.pyogenes;

differs by at least 1, 2, 5, 10 or 20 amino acids but by no more than90, 80, 70, 60, 50, 40 or 30 amino acids from amino acids 1-180 of theamino acid sequence of Cas9 of S. pyogenes, S. thermophilus, S. mutans,or Listeria innocua; or

is identical to amino acids 1-180 of the amino acid sequence of Cas9 ofS. pyogenes, S. thermophilus, S. mutans, or L. innocua.

In one embodiment, a Cas9 molecule or Cas9 polypeptide comprises anamino acid sequence referred to as region 1′:

having 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%homology with amino acids 120-180 of the amino acid sequence of Cas9 ofS. pyogenes, S. thermophilus, S. mutans or L. innocua;

differs by at least 1, 2, or 5 amino acids but by no more than 35, 30,25, 20 or 10 amino acids from amino acids 120-180 of the amino acidsequence of Cas9 of S. pyogenes, S. thermophilus, S. mutans, or L.innocua; or

is identical to amino acids 120-180 of the amino acid sequence of Cas9of S. pyogenes, S. thermophilus, S. mutans, or L. innocua.

In one embodiment, a Cas9 molecule or Cas9 polypeptide comprises anamino acid sequence referred to as region 2:

having 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%or 99% homology with amino acids 360-480 of the amino acid sequence ofCas9 of S. pyogenes, S. thermophilus, S. mutans, or L. innocua;

differs by at least 1, 2, or 5 amino acids but by no more than 35, 30,25, 20 or 10 amino acids from amino acids 360-480 of the amino acidsequence of Cas9 of S. pyogenes, S. thermophilus, S. mutans, or L.innocua; or

is identical to amino acids 360-480 of the amino acid sequence of Cas9of S. pyogenes, S. thermophilus, S. mutans, or L. innocua.

In certain embodiments, a Cas9 molecule or Cas9 polypeptide comprises anamino acid sequence referred to as region 3:

having 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or99% homology with amino acids 660-720 of the amino acid sequence of Cas9of S. pyogenes, S. thermophilus, S. mutans or L. innocua;

differs by at least 1, 2, or 5 amino acids but by no more than 35, 30,25, 20 or 10 amino acids from amino acids 660-720 of the amino acidsequence of Cas9 of S. pyogenes, S. thermophilus, S. mutans or L.innocua; or

is identical to amino acids 660-720 of the amino acid sequence of Cas9of S. pyogenes, S. thermophilus, S. mutans or L. innocua.

In one embodiment, a Cas9 molecule or Cas9 polypeptide comprises anamino acid sequence referred to as region 4:

having 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%,or 99% homology with amino acids 817-900 of the amino acid sequence ofCas9 of S. pyogenes, S. thermophilus, S. mutans, or L. innocua;

differs by at least 1, 2, or 5 amino acids but by no more than 35, 30,25, 20 or 10 amino acids from amino acids 817-900 of the amino acidsequence of Cas9 of S. pyogenes, S. thermophilus, S. mutans, or L.innocua; or

is identical to amino acids 817-900 of the amino acid sequence of Cas9of S. pyogenes, S. thermophilus, S. mutans, or L. innocua.

In one embodiment, a Cas9 molecule or Cas9 polypeptide comprises anamino acid sequence referred to as region 5:

having 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%,or 99% homology with amino acids 900-960 of the amino acid sequence ofCas9 of S. pyogenes, S. thermophilus, S. mutans, or L. innocua;

differs by at least 1, 2, or 5 amino acids but by no more than 35, 30,25, 20 or 10 amino acids from amino acids 900-960 of the amino acidsequence of Cas9 of S. pyogenes, S. thermophilus, S. mutans, or L.innocua; or

is identical to amino acids 900-960 of the amino acid sequence of Cas9of S. pyogenes, S. thermophilus, S. mutans, or L. innocua.

Engineered or Altered Cas9 Molecules and Cas9 Polypeptides

Cas9 molecules and Cas9 polypeptides described herein can possess any ofa number of properties, including: nickase activity, nuclease activity(e.g., endonuclease and/or exonuclease activity); helicase activity; theability to associate functionally with a gRNA molecule; and the abilityto target (or localize to) a site on a nucleic acid (e.g., PAMrecognition and specificity). In certain embodiments, a Cas9 molecule orCas9 polypeptide can include all or a subset of these properties. In atypical embodiment, a Cas9 molecule or Cas9 polypeptide has the abilityto interact with a gRNA molecule and, in concert with the gRNA molecule,localize to a site in a nucleic acid. Other activities, e.g., PAMspecificity, cleavage activity, or helicase activity can vary morewidely in Cas9 molecules and Cas9 polypeptides.

Cas9 molecules include engineered Cas9 molecules and engineered Cas9polypeptides (engineered, as used in this context, means merely that theCas9 molecule or Cas9 polypeptide differs from a reference sequences,and implies no process or origin limitation). An engineered Cas9molecule or Cas9 polypeptide can comprise altered enzymatic properties,e.g., altered nuclease activity, (as compared with a naturally occurringor other reference Cas9 molecule) or altered helicase activity. Asdiscussed herein, an engineered Cas9 molecule or Cas9 polypeptide canhave nickase activity (as opposed to double-strand nuclease activity).In one embodiment an engineered Cas9 molecule or Cas9 polypeptide canhave an alteration that alters its size, e.g., a deletion of amino acidsequence that reduces its size, e.g., without significant effect on oneor more, or any Cas9 activity. In one embodiment, an engineered Cas9molecule or Cas9 polypeptide can comprise an alteration that affects PAMrecognition. For example, an engineered Cas9 molecule can be altered torecognize a PAM sequence other than that recognized by the endogenouswild-type PI domain. In one embodiment a Cas9 molecule or Cas9polypeptide can differ in sequence from a naturally occurring Cas9molecule but not have significant alteration in one or more Cas9activities.

Cas9 molecules or Cas9 polypeptides with desired properties can be madein a number of ways, e.g., by alteration of a parental, e.g., naturallyoccurring, Cas9 molecules or Cas9 polypeptides, to provide an alteredCas9 molecule or Cas9 polypeptide having a desired property. Forexample, one or more mutations or differences relative to a parentalCas9 molecule, e.g., a naturally occurring or engineered Cas9 molecule,can be introduced. Such mutations and differences comprise:substitutions (e.g., conservative substitutions or substitutions ofnon-essential amino acids); insertions; or deletions. In one embodiment,a Cas9 molecule or Cas9 polypeptide can comprises one or more mutationsor differences, e.g., at least 1, 2, 3, 4, 5, 10, 15, 20, 30, 40 or 50mutations but less than 200, 100, or 80 mutations relative to areference, e.g., a parental, Cas9 molecule.

In certain embodiments, a mutation or mutations do not have asubstantial effect on a Cas9 activity, e.g., a Cas9 activity describedherein. In other embodiments, a mutation or mutations have a substantialeffect on a Cas9 activity, e.g., a Cas9 activity described herein.

Non-Cleaving and Modified-Cleavage Cas9 Molecules and Cas9 Polypeptides

In one embodiment, a Cas9 molecule or Cas9 polypeptide comprises acleavage property that differs from naturally occurring Cas9 molecules,e.g., that differs from the naturally occurring Cas9 molecule having theclosest homology. For example, a Cas9 molecule or Cas9 polypeptide candiffer from naturally occurring Cas9 molecules, e.g., a Cas9 molecule ofS. pyogenes, as follows: its ability to modulate, e.g., decreased orincreased, cleavage of a double stranded nucleic acid (endonucleaseand/or exonuclease activity), e.g., as compared to a naturally occurringCas9 molecule (e.g., a Cas9 molecule of S. pyogenes); its ability tomodulate, e.g., decreased or increased, cleavage of a single-strand of anucleic acid, e.g., a non-complementary strand of a nucleic acidmolecule or a complementary strand of a nucleic acid molecule (nickaseactivity), e.g., as compared to a naturally occurring Cas9 molecule(e.g., a Cas9 molecule of S. pyogenes); or the ability to cleave anucleic acid molecule, e.g., a double stranded or single strandednucleic acid molecule, can be eliminated.

In certain embodiments, an eaCas9 molecule or eaCas9 polypeptidecomprises one or more of the following activities: cleavage activityassociated with an N-terminal RuvC-like domain; cleavage activityassociated with an HNH-like domain; cleavage activity associated with anHNH-like domain and cleavage activity associated with an N-terminalRuvC-like domain.

In certain embodiments, an eaCas9 molecule or eaCas9 polypeptidecomprises an active, or cleavage competent, HNH-like domain (e.g., anHNH-like domain described herein) and an inactive, or cleavageincompetent, N-terminal RuvC-like domain. An exemplary inactive, orcleavage incompetent N-terminal RuvC-like domain can have a mutation ofan aspartic acid in an N-terminal RuvC-like domain, e.g., an asparticacid at position 10 of SEQ ID NO:2, e.g., can be substituted with analanine. In one embodiment, the eaCas9 molecule or eaCas9 polypeptidediffers from wild-type in the N-terminal RuvC-like domain and does notcleave the target nucleic acid, or cleaves with significantly lessefficiency, e.g., less than 20, 10, 5, 1 or 0.1% of the cleavageactivity of a reference Cas9 molecule, e.g., as measured by an assaydescribed herein. The reference Cas9 molecule can by a naturallyoccurring unmodified Cas9 molecule, e.g., a naturally occurring Cas9molecule such as a Cas9 molecule of S. pyogenes, S. aureus, or S.thermophilus. In one embodiment, the reference Cas9 molecule is thenaturally occurring Cas9 molecule having the closest sequence identityor homology.

In certain embodiments, an eaCas9 molecule or eaCas9 polypeptidecomprises an inactive, or cleavage incompetent, HNH domain and anactive, or cleavage competent, N-terminal RuvC-like domain (e.g., aRuvC-like domain described herein). Exemplary inactive, or cleavageincompetent HNH-like domains can have a mutation at one or more of: ahistidine in an HNH-like domain, for example, at position 856 of the S.pyogenes Cas9 sequence (SEQ ID NO:2), e.g., can be substituted with analanine; and one or more asparagines in an HNH-like domain, for example,at position 870 and/or 879 of the S. pyogenes Cas9 sequence (SEQ IDNO:2) e.g., can be substituted with an alanine. In one embodiment, theeaCas9 differs from wild-type in the HNH-like domain and does not cleavethe target nucleic acid, or cleaves with significantly less efficiency,e.g., less than 20, 10, 5, 1 or 0.1% of the cleavage activity of areference Cas9 molecule, e.g., as measured by an assay described herein.The reference Cas9 molecule can by a naturally occurring unmodified Cas9molecule, e.g., a naturally occurring Cas9 molecule such as a Cas9molecule of S. pyogenes, S. aureus, or S. thermophilus. In oneembodiment, the reference Cas9 molecule is the naturally occurring Cas9molecule having the closest sequence identity or homology.

In certain embodiments, exemplary Cas9 activities comprise one or moreof PAM specificity, cleavage activity, and helicase activity. Amutation(s) can be present, e.g., in: one or more RuvC domains, e.g., anN-terminal RuvC domain; an HNH domain; a region outside the RuvC domainsand the HNH domain. In one embodiment, a mutation(s) is present in aRuvC domain. In one embodiment, a mutation(s) is present in an HNHdomain. In one embodiment, mutations are present in both a RuvC domainand an HNH domain.

Exemplary mutations that may be made in the RuvC domain or HNH domainwith reference to the S. pyogenes Cas9 sequence include: D10A, E762A,H840A, N854A, N863A and/or D986A. Exemplary mutations that may be madein the RuvC domain with reference to the S. aureus Cas9 sequence includeN580A.

In one embodiment, a Cas9 molecule is an eiCas9 molecule comprising oneor more differences in a RuvC domain and/or in an HNH domain as comparedto a reference Cas9 molecule, and the eiCas9 molecule does not cleave anucleic acid, or cleaves with significantly less efficiency than doeswild type, e.g., when compared with wild type in a cleavage assay, e.g.,as described herein, cuts with less than 50, 25, 10, or 1% of areference Cas9 molecule, as measured by an assay described herein.

Whether or not a particular sequence, e.g., a substitution, may affectone or more activity, such as targeting activity, cleavage activity,etc., can be evaluated or predicted, e.g., by evaluating whether themutation is conservative. In one embodiment, a “non-essential” aminoacid residue, as used in the context of a Cas9 molecule, is a residuethat can be altered from the wild-type sequence of a Cas9 molecule,e.g., a naturally occurring Cas9 molecule, e.g., an eaCas9 molecule,without abolishing or more preferably, without substantially altering aCas9 activity (e.g., cleavage activity), whereas changing an “essential”amino acid residue results in a substantial loss of activity (e.g.,cleavage activity).

In one embodiment, a Cas9 molecule comprises a cleavage property thatdiffers from naturally occurring Cas9 molecules, e.g., that differs fromthe naturally occurring Cas9 molecule having the closest homology. Forexample, a Cas9 molecule can differ from naturally occurring Cas9molecules, e.g., a Cas9 molecule of S aureus or S. pyogenes as follows:its ability to modulate, e.g., decreased or increased, cleavage of adouble stranded break (endonuclease and/or exonuclease activity), e.g.,as compared to a naturally occurring Cas9 molecule (e.g., a Cas9molecule of S aureus or S. pyogenes); its ability to modulate, e.g.,decreased or increased, cleavage of a single-strand of a nucleic acid,e.g., a non-complimentary strand of a nucleic acid molecule or acomplementary strand of a nucleic acid molecule (nickase activity),e.g., as compared to a naturally occurring Cas9 molecule (e.g., a Cas9molecule of S aureus or S. pyogenes); or the ability to cleave a nucleicacid molecule, e.g., a double stranded or single stranded nucleic acidmolecule, can be eliminated. In certain embodiments, the nickase is S.aureus Cas9-derived nickase comprising the sequence of SEQ ID NO: 214(D10A) or SEQ ID NO: 215 (N580A) (Friedland 2015).

In certain embodiments, the altered Cas9 molecule is an eaCas9 moleculecomprising one or more of the following activities: cleavage activityassociated with a RuvC domain; cleavage activity associated with an HNHdomain; cleavage activity associated with an HNH domain and cleavageactivity associated with a RuvC domain.

In one embodiment, the altered Cas9 molecule is an eiCas9 molecule whichdoes not cleave a nucleic acid molecule (either double stranded orsingle stranded nucleic acid molecules) or cleaves a nucleic acidmolecule with significantly less efficiency, e.g., less than 20, 10, 5,1 or 0.1% of the cleavage activity of a reference Cas9 molecule, e.g.,as measured by an assay described herein. The reference Cas9 moleculecan be a naturally occurring unmodified Cas9 molecule, e.g., a naturallyoccurring Cas9 molecule such as a Cas9 molecule of S. pyogenes, S.thermophilus, S. aureus, C. jejuni or N. meningitidis. In oneembodiment, the reference Cas9 molecule is the naturally occurring Cas9molecule having the closest sequence identity or homology. In oneembodiment, the eiCas9 molecule lacks substantial cleavage activityassociated with a RuvC domain and cleavage activity associated with anHNH domain.

In certain embodiments, the altered Cas9 molecule or Cas9 polypeptide,e.g., an eaCas9 molecule or eaCas9 polypeptide, can be a fusion, e.g.,of two of more different Cas9 molecules, e.g., of two or more naturallyoccurring Cas9 molecules of different species. For example, a fragmentof a naturally occurring Cas9 molecule of one species can be fused to afragment of a Cas9 molecule of a second species. As an example, afragment of a Cas9 molecule of S. pyogenes comprising an N-terminalRuvC-like domain can be fused to a fragment of Cas9 molecule of aspecies other than S. pyogenes (e.g., S. thermophilus) comprising anHNH-like domain.

Cas9 with Altered or No PAM Recognition

Naturally-occurring Cas9 molecules can recognize specific PAM sequences,for example the PAM recognition sequences described above for, e.g., S.pyogenes, S. thermophilus, S. mutans, and S. aureus.

In certain embodiments, a Cas9 molecule or Cas9 polypeptide has the samePAM specificities as a naturally occurring Cas9 molecule. In otherembodiments, a Cas9 molecule or Cas9 polypeptide has a PAM specificitynot associated with a naturally occurring Cas9 molecule, or a PAMspecificity not associated with the naturally occurring Cas9 molecule towhich it has the closest sequence homology. For example, a naturallyoccurring Cas9 molecule can be altered, e.g., to alter PAM recognition,e.g., to alter the PAM sequence that the Cas9 molecule or Cas9polypeptide recognizes in order to decrease off-target sites and/orimprove specificity; or eliminate a PAM recognition requirement. Incertain embodiments, a Cas9 molecule or Cas9 polypeptide can be altered,e.g., to increase length of PAM recognition sequence and/or improve Cas9specificity to high level of identity (e.g., 98%, 99% or 100% matchbetween gRNA and a PAM sequence), e.g., to decrease off-target sitesand/or increase specificity. In certain embodiments, the length of thePAM recognition sequence is at least 4, 5, 6, 7, 8, 9, 10 or 15 aminoacids in length. In one embodiment, the Cas9 specificity requires atleast 90%, 95%, 96%, 97%, 98%, 99% or more homology between the gRNA andthe PAM sequence. Cas9 molecules or Cas9 polypeptides that recognizedifferent PAM sequences and/or have reduced off-target activity can begenerated using directed evolution. Exemplary methods and systems thatcan be used for directed evolution of Cas9 molecules are described (see,e.g., Esvelt 2011). Candidate Cas9 molecules can be evaluated, e.g., bymethods described below.

Size-Optimized Cas9 Molecules

Engineered Cas9 molecules and engineered Cas9 polypeptides describedherein include a Cas9 molecule or Cas9 polypeptide comprising a deletionthat reduces the size of the molecule while still retaining desired Cas9properties, e.g., essentially native conformation, Cas9 nucleaseactivity, and/or target nucleic acid molecule recognition. Providedherein are Cas9 molecules or Cas9 polypeptides comprising one or moredeletions and optionally one or more linkers, wherein a linker isdisposed between the amino acid residues that flank the deletion.Methods for identifying suitable deletions in a reference Cas9 molecule,methods for generating Cas9 molecules with a deletion and a linker, andmethods for using such Cas9 molecules will be apparent to one ofordinary skill in the art upon review of this document.

A Cas9 molecule, e.g., a S. aureus or S. pyogenes Cas9 molecule, havinga deletion is smaller, e.g., has reduced number of amino acids, than thecorresponding naturally-occurring Cas9 molecule. The smaller size of theCas9 molecules allows increased flexibility for delivery methods, andthereby increases utility for genome-editing. A Cas9 molecule cancomprise one or more deletions that do not substantially affect ordecrease the activity of the resultant Cas9 molecules described herein.Activities that are retained in the Cas9 molecules comprising a deletionas described herein include one or more of the following:

a nickase activity, i.e., the ability to cleave a single strand, e.g.,the non-complementary strand or the complementary strand, of a nucleicacid molecule; a double stranded nuclease activity, i.e., the ability tocleave both strands of a double stranded nucleic acid and create adouble stranded break, which in one embodiment is the presence of twonickase activities; an endonuclease activity; an exonuclease activity; ahelicase activity, i.e., the ability to unwind the helical structure ofa double stranded nucleic acid; and recognition activity of a nucleicacid molecule, e.g., a target nucleic acid or a gRNA molecule.

Activity of the Cas9 molecules described herein can be assessed usingthe activity assays described herein or in the art.

Identifying Regions Suitable for Deletion

Suitable regions of Cas9 molecules for deletion can be identified by avariety of methods. Naturally-occurring orthologous Cas9 molecules fromvarious bacterial species can be modeled onto the crystal structure ofS. pyogenes Cas9 (Nishimasu 2014) to examine the level of conservationacross the selected Cas9 orthologs with respect to the three-dimensionalconformation of the protein. Less conserved or unconserved regions thatare spatially located distant from regions involved in Cas9 activity,e.g., interface with the target nucleic acid molecule and/or gRNA,represent regions or domains are candidates for deletion withoutsubstantially affecting or decreasing Cas9 activity.

Nucleic Acids Encoding Cas9 Molecules

Nucleic acids encoding the Cas9 molecules or Cas9 polypeptides, e.g., aneaCas9 molecule or eaCas9 polypeptides are provided herein. Exemplarynucleic acids encoding Cas9 molecules or Cas9 polypeptides have beendescribed previously (see, e.g., Cong 2013; Wang 2013; Mali 2013; Jinek2012).

In one embodiment, a nucleic acid encoding a Cas9 molecule or Cas9polypeptide can be a synthetic nucleic acid sequence. For example, thesynthetic nucleic acid molecule can be chemically modified, e.g., asdescribed herein. In one embodiment, the Cas9 mRNA has one or more(e.g., all of the following properties: it is capped, polyadenylated,substituted with 5-methylcytidine and/or pseudouridine.

In addition, or alternatively, the synthetic nucleic acid sequence canbe codon optimized, e.g., at least one non-common codon or less-commoncodon has been replaced by a common codon. For example, the syntheticnucleic acid can direct the synthesis of an optimized messenger mRNA,e.g., optimized for expression in a mammalian expression system, e.g.,described herein.

In addition, or alternatively, a nucleic acid encoding a Cas9 moleculeor Cas9 polypeptide may comprise a nuclear localization sequence (NLS).Nuclear localization sequences are known in the art.

An exemplary codon optimized nucleic acid sequence encoding a Cas9molecule of S. pyogenes is set forth in SEQ ID NO: 22. The correspondingamino acid sequence of an S. pyogenes Cas9 molecule is set forth in SEQID NO: 23.

Exemplary codon optimized nucleic acid sequence encoding a Cas9 moleculeof S. aureus is set forth in SEQ ID NO: 26, 39, 213 and 214.

If any of the above Cas9 sequences are fused with a peptide orpolypeptide at the C-terminus, it is understood that the stop codon willbe removed.

Other Cas Molecules and Cas Polypeptides

Various types of Cas molecules or Cas polypeptides can be used topractice the inventions disclosed herein. In some embodiments, Casmolecules of Type II Cas systems are used. In other embodiments, Casmolecules of other Cas systems are used. For example, Type I or Type IIICas molecules may be used. Exemplary Cas molecules (and Cas systems)have been described previously (see, e.g., Haft 2005; Makarova 2011).Exemplary Cas molecules (and Cas systems) are also shown in Table 4.

TABLE 4 Cas Systems Structure of Families (and encoded superfamily) ofGene System type Name from protein (PDB encoded name^(‡) or subtype Haft2005^(§) accessions)^(¶) protein^(#)** Representatives cas1 Type I cas13GOD, 3LFX COG1518 SERP2463, SPy1047 Type II and 2YZS and ygbT Type IIIcas2 Type I cas2 2IVY, 2I8E and COG1343 and SERP2462, SPy1048, Type II3EXC COG3512 SPy1723 (N-terminal Type III domain) and ygbF cas 3′ TypeI^(‡‡) cas3 NA COG1203 APE1232 and ygcB cas 3″ Subtype I-A NA NA COG2254APE1231 and Subtype I-B BH0336 cas4 Subtype I-A cas4 and csa1 NA COG1468APE1239 and Subtype I-B BH0340 Subtype I-C Subtype I-D Subtype II-B cas5Subtype I-A cas5a, cas5d, 3KG4 COG1688 APE1234, BH0337, Subtype I-Bcas5e, cas5h, (RAMP) devS and ygcI Subtype I-C cas5p, cas5t Subtype I-Eand cmx5 cas6 Subtype I-A cas6 and cmx6 3I4H COG1583 and PF1131 andslr7014 Subtype I-B COG5551 Subtype I-D (RAMP) Subtype III-A SubtypeIII-B cas6e Subtype I-E cse3 1WJ9 (RAMP) ygcH cas6f Subtype I-F csy42XLJ (RAMP) y1727 cas7 Subtype I-A csa2, csd2, NA COG1857 and devR andygcJ Subtype I-B cse4, csh2, COG3649 Subtype I-C csp1 and cst2 (RAMP)Subtype I-E cas8a1 Subtype I-A^(‡‡) cmx1, cst1, NA BH0338-likeLA3191^(§§) and csx8, csx13 PG2018^(§§) and CXXC- CXXC cas8a2 SubtypeI-A^(‡‡) csa4 and csx9 NA PH0918 AF0070, AF1873, MJ0385, PF0637, PH0918and SSO1401 cas8b Subtype I-B^(‡‡) csh1 and NA BH0338-like MTH1090 andTM1802 TM1802 cas8c Subtype I-C^(‡‡) csd1 and csp2 NA BH0338-like BH0338cas9 Type II^(‡‡) csn1 and csx12 NA COG3513 FTN_0757 and SPy1046 cas10Type III^(‡‡) cmr2, csm1 NA COG1353 MTH326, Rv2823c^(§§) and csx11 andTM1794^(§§) cas10d Subtype I-D^(‡‡) csc3 NA COG1353 slr7011 csy1 SubtypeI-F^(‡‡) csy1 NA y1724-like y1724 csy2 Subtype I-F csy2 NA (RAMP) y1725csy3 Subtype I-F csy3 NA (RAMP) y1726 cse1 Subtype I-E^(‡‡) cse1 NAYgcL-like ygcL cse2 Subtype I-E cse2 2ZCA YgcK-like ygcK csc1 SubtypeI-D csc1 NA alr1563-like alr1563 (RAMP) csc2 Subtype I-D csc1 and csc2NA COG1337 slr7012 (RAMP) csa5 Subtype I-A csa5 NA AF1870 AF1870,MJ0380, PF0643 and SSO1398 csn2 Subtype II-A csn2 NA SPy1049-likeSPy1049 csm2 Subtype III-A^(‡‡) csm2 NA COG1421 MTH1081 and SERP2460csm3 Subtype III-A csc2 and csm3 NA COG1337 MTH1080 and (RAMP) SERP2459csm4 Subtype III-A csm4 NA COG1567 MTH1079 and (RAMP) SERP2458 csm5Subtype III-A csm5 NA COG1332 MTH1078 and (RAMP) SERP2457 csm6 SubtypeIII-A APE2256 and 2WTE COG1517 APE2256 and csm6 SSO1445 cmr1 SubtypeIII-B cmr1 NA COG1367 PF1130 (RAMP) cmr3 Subtype III-B cmr3 NA COG1769PF1128 (RAMP) cmr4 Subtype III-B cmr4 NA COG1336 PF1126 (RAMP) cmr5Subtype III-B^(‡‡) cmr5 2ZOP and COG3337 MTH324 and PF1125 2OEB cmr6Subtype III-B cmr6 NA COG1604 PF1124 (RAMP) csb1 Subtype I-U GSU0053 NA(RAMP) Balac_1306 and GSU0053 csb2 Subtype I-U^(‡‡) NA NA (RAMP)Balac_1305 and GSU0054 csb3 Subtype I-U NA NA (RAMP) Balac_1303^(§§)csx17 Subtype I-U NA NA NA Btus_2683 csx14 Subtype I-U NA NA NA GSU0052csx10 Subtype I-U csx10 NA (RAMP) Caur_2274 csx16 Subtype III-U VVA1548NA NA VVA1548 csaX Subtype III-U csaX NA NA SSO1438 csx3 Subtype III-Ucsx3 NA NA AF1864 csx1 Subtype III-U csa3, csx1, 1XMX and 2I71 COG1517and MJ1666, NE0113, csx2, DXTHG, COG4006 PF1127 and TM1812 NE0113 andTIGR02710 csx15 Unknown NA NA TTE2665 TTE2665 csf1 Type U csf1 NA NAAFE_1038 csf2 Type U csf2 NA (RAMP) AFE_1039 csf3 Type U csf3 NA (RAMP)AFE_1040 csf4 Type U csf4 NA NA AFE_1037

Functional Analysis of Candidate Molecules

Candidate Cas9 molecules, candidate gRNA molecules, candidate Cas9molecule/gRNA molecule complexes, can be evaluated by art-known methodsor as described herein. For example, exemplary methods for evaluatingthe endonuclease activity of Cas9 molecule have been describedpreviously (Jinek 2012).

Binding and Cleavage Assay: Testing the Endonuclease Activity of Cas9Molecule

The ability of a Cas9 molecule/gRNA molecule complex to bind to andcleave a target nucleic acid can be evaluated in a plasmid cleavageassay. In this assay, synthetic or in vitro-transcribed gRNA molecule ispre-annealed prior to the reaction by heating to 95° C. and slowlycooling down to room temperature. Native or restrictiondigest-linearized plasmid DNA (300 ng (˜8 nM)) is incubated for 60 minat 37° C. with purified Cas9 protein molecule (50-500 nM) and gRNA(50-500 nM, 1:1) in a Cas9 plasmid cleavage buffer (20 mM HEPES pH 7.5,150 mM KCl, 0.5 mM DTT, 0.1 mM EDTA) with or without 10 mM MgCl₂. Thereactions are stopped with 5×DNA loading buffer (30% glycerol, 1.2% SDS,250 mM EDTA), resolved by a 0.8 or 1% agarose gel electrophoresis andvisualized by ethidium bromide staining. The resulting cleavage productsindicate whether the Cas9 molecule cleaves both DNA strands, or only oneof the two strands. For example, linear DNA products indicate thecleavage of both DNA strands. Nicked open circular products indicatethat only one of the two strands is cleaved.

Alternatively, the ability of a Cas9 molecule/gRNA molecule complex tobind to and cleave a target nucleic acid can be evaluated in anoligonucleotide DNA cleavage assay. In this assay, DNA oligonucleotides(10 pmol) are radiolabeled by incubating with 5 units T4 polynucleotidekinase and ˜3-6 pmol (˜20-40 mCi) [γ-32P]-ATP in 1×T4 polynucleotidekinase reaction buffer at 37° C. for 30 min, in a 50 μL reaction. Afterheat inactivation (65° C. for 20 min), reactions are purified through acolumn to remove unincorporated label. Duplex substrates (100 nM) aregenerated by annealing labeled oligonucleotides with equimolar amountsof unlabeled complementary oligonucleotide at 95° C. for 3 min, followedby slow cooling to room temperature. For cleavage assays, gRNA moleculesare annealed by heating to 95° C. for 30 s, followed by slow cooling toroom temperature. Cas9 (500 nM final concentration) is pre-incubatedwith the annealed gRNA molecules (500 nM) in cleavage assay buffer (20mM HEPES pH 7.5, 100 mM KCl, 5 mM MgCl2, 1 mM DTT, 5% glycerol) in atotal volume of 9 μL. Reactions are initiated by the addition of 1 μltarget DNA (10 nM) and incubated for 1 h at 37° C. Reactions arequenched by the addition of 20 μL of loading dye (5 mM EDTA, 0.025% SDS,5% glycerol in formamide) and heated to 95° C. for 5 min. Cleavageproducts are resolved on 12% denaturing polyacrylamide gels containing 7M urea and visualized by phosphorimaging. The resulting cleavageproducts indicate that whether the complementary strand, thenon-complementary strand, or both, are cleaved.

One or both of these assays can be used to evaluate the suitability of acandidate gRNA molecule or candidate Cas9 molecule.

Binding Assay: Testing the Binding of Cas9 Molecule to Target DNA

Exemplary methods for evaluating the binding of Cas9 molecule to targetDNA have been described previously (Jinek 2012).

For example, in an electrophoretic mobility shift assay, target DNAduplexes are formed by mixing of each strand (10 nmol) in deionizedwater, heating to 95° C. for 3 min and slow cooling to room temperature.All DNAs are purified on 8% native gels containing 1× TBE. DNA bands arevisualized by UV shadowing, excised, and eluted by soaking gel pieces inDEPC-treated H2O. Eluted DNA is ethanol precipitated and dissolved inDEPC-treated H2O. DNA samples are 5′ end labeled with [γ-32P]-ATP usingT4 polynucleotide kinase for 30 min at 37° C. Polynucleotide kinase isheat denatured at 65° C. for 20 min, and unincorporated radiolabel isremoved using a column. Binding assays are performed in buffercontaining 20 mM HEPES pH 7.5, 100 mM KCl, 5 mM MgCl₂, 1 mM DTT and 10%glycerol in a total volume of 10 μL. Cas9 protein molecule is programmedwith equimolar amounts of pre-annealed gRNA molecule and titrated from100 pM to 1 μM. Radiolabeled DNA is added to a final concentration of 20pM. Samples are incubated for 1 h at 37° C. and resolved at 4° C. on an8% native polyacrylamide gel containing 1×TBE and 5 mM MgCl₂. Gels aredried and DNA visualized by phosphorimaging.

Differential Scanning Flourimetry (DSF)

The thermostability of Cas9 molecule-gRNA ribonucleoprotein (RNP)complexes can be measured via DSF. This technique measures thethermostability of a protein, which can increase under favorableconditions such as the addition of a binding RNA molecule, e.g., a gRNA.

The assay is performed using two different protocols, one to test thebest stoichiometric ratio of gRNA:Cas9 protein and another to determinethe best solution conditions for RNP formation.

To determine the best solution to form RNP complexes, a 2 μM solution ofCas9 in water+10×SYPRO Orange® (Life Technologies cat #S-6650) anddispensed into a 384 well plate. An equimolar amount of gRNA diluted insolutions with varied pH and salt is then added. After incubating atroom temperature for 10 min. and brief centrifugation to remove anybubbles, a Bio-Rad CFX384™ Real-Time System C1000 Touch™ Thermal Cyclerwith the Bio-Rad CFX Manager software is used to run a gradient from 20°C. to 90° C. with a 1° C. increase in temperature every 10 seconds.

The second assay consists of mixing various concentrations of gRNA with2 μM Cas 9 in optimal buffer from the assay above and incubating at RTfor 10 min in a 384 well plate. An equal volume of optimalbuffer+10×SYPRO Orange® (Life Technologies cat #S-6650) is added and theplate sealed with Microseal® B adhesive (MSB-1001). Following briefcentrifugation to remove any bubbles, a Bio-Rad CFX384™ Real-Time SystemC1000 Touch™ Thermal Cycler with the Bio-Rad CFX Manager software isused to run a gradient from 20° C. to 90° C. with a 1° C. increase intemperature every 10 seconds.

Resection Assay: Testing a Cas9 to Promote Resection

The ability of a Cas9 to promote resection can be evaluated by measuringthe levels of single stranded DNA at specific double strand break sitesin human cells using quantitative methods (as described in Zhou 2014).In this assay, a cell line is delivered, e.g., by transfection, acandidate Cas9 or a candidate Cas9 fusion protein. The cells arecultured for a sufficient amount of time to allow nuclease activity andresection to occur. Genomic DNA is carefully extracted using a method inwhich cells are embedded in low-gelling point agar that protects the DNAfrom shearing and damage during extraction. The genomic DNA is digestedwith a restriction enzyme that selectively cuts double-stranded DNA.Primers for quantitative PCR that span up to 5 kb of the double strandbreak site are designed. The results from the PCR reaction show thelevels of single strand DNA detected at each of the primer positions.Thus, the length and the level of resection promoted by the candidateCas9 or Cas9 fusion protein can be determined from this assay.

Other qualitative assays for identifying the occurrence of resectioninclude the detection of proteins or protein complexes that bind tosingle-stranded DNA after resection has occurred, e.g., RPA foci, Rad51foci, or BrDU detection by immunofluorescence. Antibodies for RPAprotein and Rad51 are known in the art.

Genome Editing Approaches

Mutations in a target gene may be corrected using one of the approachesdiscussed herein. In one embodiment, a mutation in a target gene iscorrected by homology directed repair (HDR) using an exogenouslyprovided template nucleic acid, referred to herein as “gene correction”.In another embodiment, a mutation in a target gene is corrected byhomology directed repair without using an exogenously provided templatenucleic acid, referred to herein as gene correction.

HDR Repair and Template Nucleic Acids

In certain embodiments of the methods provided herein, HDR-mediatedsequence alteration is used to alter and/or correct (e.g., repair oredit) the sequence of one or more nucleotides in a genome (e.g., a pointmutation in a target gene). While not wishing to be bound by theory, itis believed that HDR-mediated alteration of a target sequence within atarget gene occurs by HDR with an exogenously provided donor template ortemplate nucleic acid in a process referred to herein as genecorrection. For example, the donor template or template nucleic acidprovides for alteration of the target sequence. It is contemplated thata plasmid donor can be used as a template for homologous recombination.It is further contemplated that a single stranded donor template can beused as a template for alteration of the target sequence by alternatemethods of HDR (e.g., single-strand annealing) between the targetsequence and the donor template. Donor template-effected alteration of atarget sequence depends on cleavage by a Cas9 molecule. Cleavage by Cas9can comprise a double-strand break or two single-strand breaks.

In one embodiment, the target position or target position regions has atleast 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology with anendogenous homologous sequence.

In one embodiment, the target position region, except for the targetposition, differs by 1, 2, 3, 4, 5, 10, 25, 50, 100 or fewer,nucleotides with an endogenous homologous sequence.

In one embodiment, the target position region has at least 50%, 60%,70%, 80%, 90%, 92%, 94%, 96%, 98%, or 99% homology with an endogenoushomologous sequence over at least 10, 20, 30, 40, 50, 100, 200, 300,400, 500, 750, 1,000, 2500, 5000, or 10000 nucleotides.

In one embodiment, the target position region, except for the targetposition, differs by 1, 2, 3, 4, 5, 10, 25, 50, 100 or fewer,nucleotides with an endogenous homologous sequence over at least 10, 20,30, 40, 50, 100, 200, 300, 400, 500, 750, 1,000, 2500, 5000, or 10000nucleotides.

In one embodiment, the endogenous homologous sequence comprises adomain, e.g., a catalytic domain, a domain that binds a target, astructural domain, found in the gene that comprises the target position.

In certain embodiments of the methods provided herein, HDR-mediatedalteration is used to alter a single nucleotide in a target sequence.These embodiments may utilize either one double-strand break or twosingle-strand breaks. In certain embodiments, a single nucleotidealteration is incorporated using (1) one double-strand break, (2) twosingle-strand breaks, (3) two double-strand breaks with a breakoccurring on each side of the target position, (4) one double-strandbreak and two single-strand breaks with the double-strand break and twosingle-strand breaks occurring on each side of the target position (5)four single-strand breaks with a pair of single stranded breaksoccurring on each side of the target position, or (6) one single-strandbreak.

In certain embodiments wherein a single-stranded template nucleic acidis used, the target position can be altered by alternative HDR.

Donor template-effected alteration of a target position depends oncleavage by a Cas9 molecule. Cleavage by Cas9 can comprise a nick, adouble-strand break, or two single-strand breaks, e.g., one on eachstrand of the target nucleic acid. After introduction of the breaks onthe target nucleic acid, resection occurs at the break ends resulting insingle stranded overhanging DNA regions.

In canonical HDR, a double-stranded donor template is introduced,comprising homologous sequence to the target nucleic acid that willeither be directly incorporated into the target nucleic acid or used asa template to change the sequence of the target nucleic acid. Afterresection at the break, repair can progress by different pathways, e.g.,by the double Holliday junction model (or double-strand break repair,DSBR, pathway) or the synthesis-dependent strand annealing (SDSA)pathway. In the double Holliday junction model, strand invasion by thetwo single stranded overhangs of the target nucleic acid to thehomologous sequences in the donor template occurs, resulting in theformation of an intermediate with two Holliday junctions. The junctionsmigrate as new DNA is synthesized from the ends of the invading strandto fill the gap resulting from the resection. The end of the newlysynthesized DNA is ligated to the resected end, and the junctions areresolved, resulting in the alteration of the target nucleic acid, e.g.,incorporation of the altered sequence of the donor template at thecorresponding target position. Crossover with the donor template mayoccur upon resolution of the junctions. In the SDSA pathway, only onesingle stranded overhang invades the donor template and new DNA issynthesized from the end of the invading strand to fill the gapresulting from resection. The newly synthesized DNA then anneals to theremaining single stranded overhang, new DNA is synthesized to fill inthe gap, and the strands are ligated to produce the altered DNA duplex.

In alternative HDR, a single-strand donor template, e.g., templatenucleic acid, is introduced. A nick, single-strand break, ordouble-strand break at the target nucleic acid, for altering a desiredtarget position, is mediated by a Cas9 molecule, e.g., described herein,and resection at the break occurs to reveal single stranded overhangs.Incorporation of the sequence of the template nucleic acid to correct oralter the target position of the target nucleic acid typically occurs bythe SDSA pathway, as described above.

Additional details on template nucleic acids are provided in Section IVentitled “Template nucleic acids” in International ApplicationPCT/US2014/057905, now published as WO2015/048577, the entire contentsof which are expressly incorporated herein by reference.

In certain embodiments, double-strand cleavage is effected by a Cas9molecule having cleavage activity associated with an HNH-like domain andcleavage activity associated with a RuvC-like domain, e.g., anN-terminal RuvC-like domain, e.g., a wild type Cas9. Such embodimentsrequire only a single gRNA molecule.

In certain embodiments, one single-strand break, or nick, is effected bya Cas9 molecule having nickase activity, e.g., a Cas9 nickase asdescribed herein. A nicked target nucleic acid can be a substrate foralt-HDR.

In other embodiments, two single-strand breaks, or nicks, are effectedby a Cas9 molecule having nickase activity, e.g., cleavage activityassociated with an HNH-like domain or cleavage activity associated withan N-terminal RuvC-like domain. Such embodiments usually require twogRNAs, one for placement of each single-strand break. In one embodiment,the Cas9 molecule having nickase activity cleaves the strand to whichthe gRNA hybridizes, but not the strand that is complementary to thestrand to which the gRNA hybridizes. In one embodiment, the Cas9molecule having nickase activity does not cleave the strand to which thegRNA hybridizes, but rather cleaves the strand that is complementary tothe strand to which the gRNA hybridizes.

In certain embodiments, the nickase has HNH activity, e.g., a Cas9molecule having the RuvC activity inactivated, e.g., a Cas9 moleculehaving a mutation at D10, e.g., the D10A mutation. D10A inactivatesRuvC; therefore, the Cas9 nickase has (only) HNH activity and will cuton the strand to which the gRNA hybridizes (e.g., the complementarystrand, which does not have the NGG PAM on it). In other embodiments, aCas9 molecule having an H840, e.g., an H840A, mutation can be used as anickase. H840A inactivates HNH; therefore, the Cas9 nickase has (only)RuvC activity and cuts on the non-complementary strand (e.g., the strandthat has the NGG PAM and whose sequence is identical to the gRNA). Inother embodiments, a Cas9 molecule having an N863 mutation, e.g., theN863A mutation, mutation can be used as a nickase. N863A inactivates HNHtherefore the Cas9 nickase has (only) RuvC activity and cuts on thenon-complementary strand (the strand that has the NGG PAM and whosesequence is identical to the gRNA).

In certain embodiments, in which a nickase and two gRNAs are used toposition two single-strand nicks, one nick is on the + strand and onenick is on the − strand of the target nucleic acid. The PAMs can beoutwardly facing or inwardly facing. The gRNAs can be selected such thatthe gRNAs are separated by, from about 0-50, 0-100, or 0-200nucleotides. In one embodiment, there is no overlap between the targetsequences that are complementary to the targeting domains of the twogRNAs. In one embodiment, the gRNAs do not overlap and are separated byas much as 50, 100, or 200 nucleotides. In one embodiment, the use oftwo gRNAs can increase specificity, e.g., by decreasing off-targetbinding (Ran 2013).

In certain embodiments, a single nick can be used to induce HDR, e.g.,alt-HDR. It is contemplated herein that a single nick can be used toincrease the ratio of HR to NHEJ at a given cleavage site. In certainembodiments, a single-strand break is formed in the strand of the targetnucleic acid to which the targeting domain of said gRNA iscomplementary. In certain embodiments, a single-strand break is formedin the strand of the target nucleic acid other than the strand to whichthe targeting domain of said gRNA is complementary.

Placement of Double-Strand or Single-Strand Breaks Relative to theTarget Position

A double-strand break or single-strand break in one of the strandsshould be sufficiently close to target position that an alteration isproduced in the desired region, e.g., correction of a mutation occurs.In certain embodiments, the distance is not more than 50, 100, 200, 300,350 or 400 nucleotides. While not wishing to be bound by theory, incertain embodiments, it is believed that the break should besufficiently close to target position such that the target position iswithin the region that is subject to exonuclease-mediated removal duringend resection. If the distance between the target position and a breakis too great, the sequence desired to be altered may not be included inthe end resection and, therefore, may not be altered, as donor sequence,either exogenously provided donor sequence or endogenous genomic donorsequence, in some embodiments is only used to alter sequence within theend resection region.

In certain embodiments, the gRNA targeting domain is configured suchthat a cleavage event, e.g., a double-strand or single-strand break, ispositioned within 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60,70, 80, 90, 100, 150 or 200 nucleotides of the region desired to bealtered, e.g., a mutation. The break, e.g., a double-strand orsingle-strand break, can be positioned upstream or downstream of theregion desired to be altered, e.g., a mutation. In some embodiments, abreak is positioned within the region desired to be altered, e.g.,within a region defined by at least two mutant nucleotides. In someembodiments, a break is positioned immediately adjacent to the regiondesired to be altered, e.g., immediately upstream or downstream of amutation.

In certain embodiments, a single-strand break is accompanied by anadditional single-strand break, positioned by a second gRNA molecule, asdiscussed below. For example, the targeting domains bind configured suchthat a cleavage event, e.g., the two single-strand breaks, arepositioned within 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60,70, 80, 90, 100, 150 or 200 nucleotides of a target position. In oneembodiment, the first and second gRNA molecules are configured such thatwhen guiding a Cas9 nickase, a single-strand break is accompanied by anadditional single-strand break, positioned by a second gRNA,sufficiently close to one another to result in alteration of the desiredregion. In one embodiment, the first and second gRNA molecules areconfigured such that a single-strand break positioned by said secondgRNA is within 10, 20, 30, 40, or 50 nucleotides of the break positionedby said first gRNA molecule, e.g., when the Cas9 is a nickase. In oneembodiment, the two gRNA molecules are configured to position cuts atthe same position, or within a few nucleotides of one another, ondifferent strands, e.g., essentially mimicking a double-strand break.

In certain embodiments, in which a gRNA (unimolecular (or chimeric) ormodular gRNA) and Cas9 nuclease induce a double-strand break for thepurpose of inducing HDR-mediated alteration, the cleavage site isbetween 0-200 bp (e.g., 0-175, 0 to 150, 0 to 125, 0 to 100, 0 to 75, 0to 50, 0 to 25, 25 to 200, 25 to 175, 25 to 150, 25 to 125, 25 to 100,25 to 75, 25 to 50, 50 to 200, 50 to 175, 50 to 150, 50 to 125, 50 to100, 50 to 75, 75 to 200, 75 to 175, 75 to 150, 75 to 125, 75 to 100 bp)away from the target position. In certain embodiments, the cleavage siteis between 0-100 bp (e.g., 0 to 75, 0 to 50, 0 to 25, 25 to 100, 25 to75, 25 to 50, 50 to 100, 50 to 75 or 75 to 100 bp) away from the targetposition.

In certain embodiments, one can promote HDR by using nickases togenerate a break with overhangs. While not wishing to be bound bytheory, the single stranded nature of the overhangs can enhance thecell's likelihood of repairing the break by HDR as opposed to, e.g.,NHEJ. Specifically, in certain embodiments, HDR is promoted by selectinga first gRNA that targets a first nickase to a first target sequence,and a second gRNA that targets a second nickase to a second targetsequence which is on the opposite DNA strand from the first targetsequence and offset from the first nick.

In certain embodiment, the targeting domain of a gRNA molecule isconfigured to position a cleavage event sufficiently far from apreselected nucleotide, e.g., the nucleotide of a coding region, suchthat the nucleotide is not altered. In certain embodiments, thetargeting domain of a gRNA molecule is configured to position anintronic cleavage event sufficiently far from an intron/exon border, ornaturally occurring splice signal, to avoid alteration of the exonicsequence or unwanted splicing events. The gRNA molecule may be a first,second, third and/or fourth gRNA molecule, as described herein.

Placement of a First Break and a Second Break Relative to Each Other

In certain embodiments, a double-strand break can be accompanied by anadditional double-strand break, positioned by a second gRNA molecule, asis discussed below.

In certain embodiments, a double-strand break can be accompanied by twoadditional single-strand breaks, positioned by a second gRNA moleculeand a third gRNA molecule.

In certain embodiments, a first and second single-strand breaks can beaccompanied by two additional single-strand breaks positioned by a thirdgRNA molecule and a fourth gRNA molecule.

When two or more gRNAs are used to position two or more cleavage events,e.g., double-strand or single-strand breaks, in a target nucleic acid,it is contemplated that the two or more cleavage events may be made bythe same or different Cas9 proteins. For example, when two gRNAs areused to position two double stranded breaks, a single Cas9 nuclease maybe used to create both double stranded breaks. When two or more gRNAsare used to position two or more single stranded breaks (nicks), asingle Cas9 nickase may be used to create the two or more nicks. Whentwo or more gRNAs are used to position at least one double strandedbreak and at least one single stranded break, two Cas9 proteins may beused, e.g., one Cas9 nuclease and one Cas9 nickase. It is contemplatedthat when two or more Cas9 proteins are used that the two or more Cas9proteins may be delivered sequentially to control specificity of adouble stranded versus a single stranded break at the desired positionin the target nucleic acid.

In some embodiments, the targeting domain of the first gRNA molecule andthe targeting domain of the second gRNA molecules are complementary toopposite strands of the target nucleic acid molecule. In someembodiments, the gRNA molecule and the second gRNA molecule areconfigured such that the PAMs are oriented outward. In some embodiments,the gRNA molecule and the second gRNA molecule are configured such thatthe PAMs are oriented inward.

In certain embodiments, two gRNA are selected to direct Cas9-mediatedcleavage at two positions that are a preselected distance from eachother. In certain embodiments, the two points of cleavage are onopposite strands of the target nucleic acid. In some embodiments, thetwo cleavage points form a blunt ended break, and in other embodiments,they are offset so that the DNA ends comprise one or two overhangs(e.g., one or more 5′ overhangs and/or one or more 3′ overhangs). Insome embodiments, each cleavage event is a nick. In some embodiments,the nicks are close enough together that they form a break that isrecognized by the double stranded break machinery (as opposed to beingrecognized by, e.g., the SSBr machinery). In certain embodiments, thenicks are far enough apart that they create an overhang that is asubstrate for HDR, i.e., the placement of the breaks mimics a DNAsubstrate that has experienced some resection. For instance, in someembodiments the nicks are spaced to create an overhang that is asubstrate for processive resection. In some embodiments, the two breaksare spaced within 25-65 nucleotides of each other. The two breaks maybe, e.g., about 25, 30, 35, 40, 45, 50, 55, 60 or 65 nucleotides of eachother. The two breaks may be, e.g., at least about 25, 30, 35, 40, 45,50, 55, 60 or 65 nucleotides of each other. The two breaks may be, e.g.,at most about 30, 35, 40, 45, 50, 55, 60 or 65 nucleotides of eachother. In embodiments, the two breaks are about 25-30, 30-35, 35-40,40-45, 45-50, 50-55, 55-60, or 60-65 nucleotides of each other.

In some embodiments, the break that mimics a resected break comprises a3′ overhang (e.g., generated by a DSB and a nick, where the nick leavesa 3′ overhang), a 5′ overhang (e.g., generated by a DSB and a nick,where the nick leaves a 5′ overhang), a 3′ and a 5′ overhang (e.g.,generated by three cuts), two 3′ overhangs (e.g., generated by two nicksthat are offset from each other), or two 5′ overhangs (e.g., generatedby two nicks that are offset from each other).

In certain embodiments, in which two gRNAs (independently, unimolecular(or chimeric) or modular gRNA) complexing with Cas9 nickases induce twosingle-strand breaks for the purpose of inducing HDR-mediated alteration(e.g., correction), the closer nick is between 0-200 bp (e.g., 0-175, 0to 150, 0 to 125, 0 to 100, 0 to 75, 0 to 50, 0 to 25, 25 to 200, 25 to175, 25 to 150, 25 to 125, 25 to 100, 25 to 75, 25 to 50, 50 to 200, 50to 175, 50 to 150, 50 to 125, 50 to 100, 50 to 75, 75 to 200, 75 to 175,75 to 150, 75 to 125, or 75 to 100 bp) away from the target position andthe two nicks will ideally be within 25-65 bp of each other (e.g., 25 to50, 25 to 45, 25 to 40, 25 to 35, 25 to 30, 30 to 55, 30 to 50, 30 to45, 30 to 40, 30 to 35, 35 to 55, 35 to 50, 35 to 45, 35 to 40, 40 to55, 40 to 50, 40 to 45 bp, 45 to 50 bp, 50 to 55 bp, 55 to 60 bp, or 60to 65 bp) and no more than 100 bp away from each other (e.g., no morethan 90, 80, 70, 60, 50, 40, 30, 20, 10, or 5 bp away from each other).In certain embodiments, the cleavage site is between 0-100 bp (e.g., 0to 75, 0 to 50, 0 to 25, 25 to 100, 25 to 75, 25 to 50, 50 to 100, 50 to75, or 75 to 100 bp) away from the target position.

In some embodiments, two gRNAs, e.g., independently, unimolecular (orchimeric) or modular gRNA, are configured to position a double-strandbreak on both sides of a target position. In other embodiments, threegRNAs, e.g., independently, unimolecular (or chimeric) or modular gRNA,are configured to position a double-strand break (i.e., one gRNAcomplexes with a Cas9 nuclease) and two single-strand breaks or pairedsingle stranded breaks (i.e., two gRNAs complex with Cas9 nickases) oneither side of the target position. In other embodiments, four gRNAs,e.g., independently, unimolecular (or chimeric) or modular gRNA, areconfigured to generate two pairs of single stranded breaks (i.e., twopairs of two gRNA molecules complex with Cas9 nickases) on either sideof the target position. The double-strand break(s) or the closer of thetwo single-strand nicks in a pair will ideally be within 0-500 bp of thetarget position (e.g., no more than 450, 400, 350, 300, 250, 200, 150,100, 50 or 25 bp from the target position). When nickases are used, thetwo nicks in a pair are, in certain embodiments, within 25-65 bp of eachother (e.g., between 25 to 55, 25 to 50, 25 to 45, 25 to 40, 25 to 35,25 to 30, 50 to 55, 45 to 55, 40 to 55, 35 to 55, 30 to 55, 30 to 50, 35to 50, 40 to 50, 45 to 50, 35 to 45, 40 to 45 bp, 45 to 50 bp, 50 to 55bp, 55 to 60 bp, or 60 to 65 bp) and no more than 100 bp away from eachother (e.g., no more than 90, 80, 70, 60, 50, 40, 30, 20, or 10 bp).

When two gRNAs are used to target Cas9 molecules to breaks, differentcombinations of Cas9 molecules are envisioned. In some embodiments, afirst gRNA is used to target a first Cas9 molecule to a first targetposition, and a second gRNA is used to target a second Cas9 molecule toa second target position. In some embodiments, the first Cas9 moleculecreates a nick on the first strand of the target nucleic acid, and thesecond Cas9 molecule creates a nick on the opposite strand, resulting ina double stranded break (e.g., a blunt ended cut or a cut withoverhangs).

Different combinations of nickases can be chosen to target one singlestranded break to one strand and a second single stranded break to theopposite strand. When choosing a combination, one can take into accountthat there are nickases having one active RuvC-like domain, and nickaseshaving one active HNH domain. In certain embodiments, a RuvC-like domaincleaves the non-complementary strand of the target nucleic acidmolecule. In certain embodiments, an HNH-like domain cleaves a singlestranded complementary domain, e.g., a complementary strand of a doublestranded nucleic acid molecule. Generally, if both Cas9 molecules havethe same active domain (e.g., both have an active RuvC domain or bothhave an active HNH domain), one will choose two gRNAs that bind toopposite strands of the target. In more detail, in some embodiments, afirst gRNA is complementary with a first strand of the target nucleicacid and binds a nickase having an active RuvC-like domain and causesthat nickase to cleave the strand that is non-complementary to thatfirst gRNA, i.e., a second strand of the target nucleic acid; and asecond gRNA is complementary with a second strand of the target nucleicacid and binds a nickase having an active RuvC-like domain and causesthat nickase to cleave the strand that is non-complementary to thatsecond gRNA, i.e., the first strand of the target nucleic acid.Conversely, In some embodiments, a first gRNA is complementary with afirst strand of the target nucleic acid and binds a nickase having anactive HNH domain and causes that nickase to cleave the strand that iscomplementary to that first gRNA, i.e., a first strand of the targetnucleic acid; and a second gRNA is complementary with a second strand ofthe target nucleic acid and binds a nickase having an active HNH domainand causes that nickase to cleave the strand that is complementary tothat second gRNA, i.e., the second strand of the target nucleic acid. Inanother arrangement, if one Cas9 molecule has an active RuvC-like domainand the other Cas9 molecule has an active HNH domain, the gRNAs for bothCas9 molecules can be complementary to the same strand of the targetnucleic acid, so that the Cas9 molecule with the active RuvC-like domainwill cleave the non-complementary strand and the Cas9 molecule with theHNH domain will cleave the complementary strand, resulting in a doublestranded break.

In one embodiment, the cleavage event comprises one or more breaks,e.g., one or more single-strand breaks, one or more double-strandbreaks, or a combination thereof.

In one embodiment, the cleavage event comprises any one of thefollowing: (a) one single-strand break; (b) two single-strand breaks;(c) three single-strand breaks; (d) four single-strand breaks; (e) onedouble-strand break; (f) two double-strand breaks; (g) one single-strandbreak and one double-strand break; (h) two single-strand breaks and onedouble-strand break; or (i) any combination thereof.

In one embodiment, the gRNA molecule and the second gRNA moleculeposition a cleavage event on each strand of a target nucleic acid.

In one embodiment, the cleavage event flanks the target position, andwherein the terminus (created by the cleavage event) closest to thetarget position, for each cleavage event, is a 5′ terminus, e.g.,resulting in a 5′ overhang.

While not wishing to be bound by theory, it believed that, in oneembodiment, the sequence exposed by a cleavage event (e.g., asingle-strand cleavage event) mediated by a gRNA molecule and a Cas9fusion molecule (e.g., a Cas9 nickase, e.g., a Cas9 molecule containingD10A or N863A mutation) may affect (e.g., increase or decrease) genecorrection efficiency. For example, the sequence exposed by the cleavageevent can include a 5′ overhang, a 3′ overhang, a product of thenucleolytic processing of a 5′ overhang, a product of the nucleolyticprocessing of a 3′ overhang, or any combination thereof. In oneembodiment, the exposed sequence comprises or consists of a 5′ overhang.In another embodiment, the exposed sequence comprises or consists of a3′ overhang. In one embodiment, the exposed sequence comprises orconsists of a product of the nucleolytic processing of a 5′ overhang. Inanother embodiment, the exposed sequence comprises or consists of aproduct of the nucleolytic processing of a 3′ overhang.

In one embodiment, the 5′ overhang is between 1 and 20000 nucleotides, 5and 20000 nucleotides, 10 and 20000 nucleotides, 20 and 20000nucleotides, 30 and 20000 nucleotides, between 35 and 20000 nucleotides,between 40 and 20000 nucleotides, between 50 and 20000 nucleotides,between 1000 and 10000 nucleotides, or between 500 and 5000 nucleotidesin length, e.g., between 1 and 100 nucleotides, between 1 and 50nucleotides, between 1 and 25 nucleotides, between 40 and 60nucleotides, between 40 and 55 nucleotides, or between 45 and 50nucleotides in length, e.g., at least about 1, 5, 10, 20, 30, 35, 40,45, 50, 75, 100, 200, 300, 400, 500, 750, 1000, 2000, 3000, 4000, 5000,6000, 7000, 8000, 9000, 10000, or 15000 nucleotides in length. Thesequence exposed by the Cas9 fusion molecule/gRNA molecule mediatedcleavage event can constitute a substrate used for homology search ingene correction.

In one embodiment, the exposed sequence differs by 1, 2, 3, 4, 5, 10,25, 50, 100 or fewer, nucleotides with an endogenous homologoussequence. In one embodiment, the exposed sequence has at least 50%, 60%,70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% homology with an endogenoushomologous sequence over at least 10, 20, 30, 40, 50, 100, 200, 300,400, 500, 750, 1000, 2500, 5000, or 10000 nucleotides. In oneembodiment, the exposed sequence differs by 1, 2, 3, 4, 5, 10, 25, 50,100 or fewer, nucleotides with an endogenous homologous sequence over atleast 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 750, 1000, 2500,5000, or 10000 nucleotides.

In one embodiment, the cleavage event flanks the target position, andthe terminus (created by a cleavage event) closest to the targetposition, for each cleavage event, is a 3′ terminus, e.g., resulting a3′ overhang.

In one embodiment, the 3′ overhang is between 1 and 20000 nucleotides, 5and 20000 nucleotides, 10 and 20000 nucleotides, 20 and 20000nucleotides, between 30 and 20000 nucleotides, between 35 and 20000nucleotides, between 40 and 20000 nucleotides, between 50 and 20000nucleotides, between 1000 and 10000 nucleotides, or between 500 and 5000nucleotides in length, e.g., between 1 and 100 nucleotides, between 1and 50 nucleotides, between 1 and 25 nucleotides, between 40 and 60nucleotides, between 40 and 55 nucleotides, or between 45 and 50nucleotides in length, e.g., at least about 30, 35, 40, 45, 50, 75, 100,200, 300, 400, 500, 750, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000,9000, 10000, or 15000 nucleotides in length.

In one embodiment, the distance between the cleavage event and thetarget position is between 10 and 10000 nucleotides in length, e.g.,between 50 and 5000 nucleotides, between 100 and 1000 nucleotides,between 200 and 800 nucleotides, between 400 and 600 nucleotides,between 100 and 500 nucleotides, or between 500 and 1000 nucleotides inlength, e.g., at least about 20, 30, 40, 50, 75, 100, 200, 300, 400,500, 750, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10000nucleotides in length.

In one embodiment, the cleavage event comprises a single-strand break,and wherein the distance between the single-strand break and the targetposition is between 10 and 10000 nucleotides in length, e.g., between 50and 5000 nucleotides, between 100 and 1000 nucleotides, between 200 and800 nucleotides, between 400 and 600 nucleotides, between 100 and 500nucleotides, or between 500 and 1000 nucleotides in length, e.g., atleast about 20, 30, 40, 50, 75, 100, 200, 300, 400, 500, 750, 1000,2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10000 nucleotides inlength.

In one embodiment, the cleavage event comprises two, three or foursingle-strand breaks, and wherein the distance between each of thesingle-strand breaks and the target position is between 10 and 10000nucleotides in length, e.g., between 50 and 5000 nucleotides, between100 and 1000 nucleotides, between 200 and 800 nucleotides, between 400and 600 nucleotides, between 100 and 500 nucleotides, or between 500 and1000 nucleotides in length, e.g., at least about 20, 30, 40, 50, 75,100, 200, 300, 400, 500, 750, 1000, 2000, 3000, 4000, 5000, 6000, 7000,8000, 9000, or 10000 nucleotides in length.

In one embodiment, the cleavage event comprises a double-strand break,and wherein the distance between the double-strand break and the targetposition is between 10 and 10000 nucleotides in length, e.g., between 50and 5000 nucleotides, between 100 and 1000 nucleotides, between 200 and800 nucleotides, between 400 and 600 nucleotides, between 100 and 500nucleotides, or between 500 and 1000 nucleotides in length, e.g., atleast about 20, 30, 40, 50, 75, 100, 200, 300, 400, 500, 750, 1000,2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10000 nucleotides inlength.

In one embodiment, the cleavage event comprises two double-strandbreaks, and wherein the distance between each of the double-strandbreaks and the target position is between 10 and 10000 nucleotides inlength, e.g., between 50 and 5000 nucleotides, between 100 and 1000nucleotides, between 200 and 800 nucleotides, between 400 and 600nucleotides, between 100 and 500 nucleotides, or between 500 and 1000nucleotides in length, e.g., at least about 20, 30, 40, 50, 75, 100,200, 300, 400, 500, 750, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000,9000, or 10000 nucleotides in length.

In one embodiment, the cleavage event comprises a single-strand breakand a double-strand break, wherein the distance between thesingle-strand break and the target position is between 10 and 10000nucleotides in length, e.g., between 50 and 5000 nucleotides or between100 and 1000 nucleotides in length, e.g., about 20, 30, 40, 50, 75, 100,200, 300, 400, 500, 750, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000,9000, or 10000 nucleotides in length, and

wherein the distance between the double-strand break and the targetposition is between 10 and 10000 nucleotides in length, e.g., between 50and 5000 nucleotides or between 100 and 1000 nucleotides in length,e.g., at least about 20, 30, 40, 50, 75, 100, 200, 300, 400, 500, 750,1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10000nucleotides in length.

In one embodiment, the cleavage event comprises two single-strand breaksand a double-strand break,

wherein the distance between each of the single-strand breaks and thetarget position is between 10 and 10000 nucleotides in length, e.g.,between 50 and 5000 nucleotides or between 100 and 1000 nucleotides inlength, e.g., about 20, 30, 40, 50, 75, 100, 200, 300, 400, 500, 750,1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10000nucleotides in length, and

wherein the distance between the double-strand break and the targetposition is between 10 and 10000 nucleotides in length, e.g., between 50and 5000 nucleotides or between 100 and 1000 nucleotides in length,e.g., at least about 20, 30, 40, 50, 75, 100, 200, 300, 400, 500, 750,1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10000nucleotides in length.

In one embodiment, the cleavage event comprises two or moresingle-strand breaks, two or more double-strand breaks, or twosingle-strand breaks and one double-strand breaks,

wherein the distance between any of the two breaks that are present onthe same strand is between 30 and 20000 nucleotides, 40 and 20000nucleotides, or 50 and 20000 nucleotides in length, e.g., between 1000and 10000 nucleotides or between 500 and 5000 nucleotides in length,e.g., between 40 and 60 nucleotides, between 40 and 55 nucleotides, orbetween 45 and 50 nucleotides in length, e.g., at least about 30, 35,40, 45, 50, 75, 100, 200, 300, 400, 500, 750, 1000, 2000, 3000, 4000,5000, 6000, 7000, 8000, 9000, 10000, or 15000 nucleotides in length.

In one embodiment, the cleavage event comprises two or moresingle-strand breaks, two or more double-strand breaks, or twosingle-strand breaks and one double-strand breaks,

wherein the distance between at least two breaks that are present ondifferent strands is between 30 and 20000 nucleotides, 40 and 20000nucleotides, or 50 and 20000 nucleotides in length, e.g., between 1000and 10000 nucleotides or between 500 and 5000 nucleotides in length,e.g., between 40 and 60 nucleotides, between 40 and 55 nucleotides, orbetween 45 and 50 nucleotides in length, e.g., at least about 30, 35,40, 45, 50, 75, 100, 200, 300, 400, 500, 750, 1000, 2000, 3000, 4000,5000, 6000, 7000, 8000, 9000, 10000, or 15000 nucleotides in length.

In one embodiment, the cleavage event comprises two single-strandbreaks, wherein the distance between the two single breaks is between 30and 20000 nucleotides, 40 and 20000 nucleotides, or 50 and 20000nucleotides in length, e.g., between 1000 and 10000 nucleotides orbetween 500 and 5000 nucleotides in length, e.g., between 40 and 60nucleotides, between 40 and 55 nucleotides, or between 45 and 50nucleotides in length, e.g., at least about 30, 35, 40, 45, 50, 75, 100,200, 300, 400, 500, 750, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000,9000, 10000, or 15000 nucleotides in length. In one embodiment, thesingle-strand breaks are present on different strands. In anotherembodiment, the single-strand breaks are present on the same strand. Inone embodiment, the cleavage event further comprises one or more (e.g.,two) of single-strand break, double-strand break, or both.

In one embodiment, the Cas9 molecule comprises HNH-like domain cleavageactivity but has no, or no significant, N-terminal RuvC-like domaincleavage activity. In one embodiment, the eaCas9 molecule is an HNH-likedomain nickase, e.g., the Cas9 molecule comprises a mutation at D10,e.g., D10A. In another embodiment, the eaCas9 molecule comprisesN-terminal RuvC-like domain cleavage activity but has no, or nosignificant, HNH-like domain cleavage activity. In one embodiment, theCas9 molecule is an N-terminal RuvC-like domain nickase, e.g., theeaCas9 molecule comprises a mutation at N863, e.g., N863A.

In one embodiment, the first gRNA molecule positions a cleavage event ona strand that does not bind to the first gRNA molecule.

In one embodiment, the second gRNA molecule positions a cleavage eventon a strand that does not bind to the second gRNA molecule.

In one embodiment, the first gRNA molecule positions a cleavage event ona strand that does not bind to the first gRNA and the second gRNAmolecule positions a cleavage event on a strand that does not bind tothe second gRNA molecule, and wherein the gRNA molecule and the secondgRNA molecule bind to different strands, e.g., resulting in a 3′overhang on each strand.

In one embodiment, the first gRNA molecule positions a cleavage event 5′to the target position on the first strand. In one embodiment, thesecond gRNA molecule positions a cleavage event 3′ to the targetposition (relative to the target position on the first strand) on thesecond strand. In one embodiment, the second gRNA molecule positions acleavage event 5′ to the target position on the second strand. In oneembodiment, the first gRNA molecule positions a cleavage event 5′ to thetarget position on the first strand, and wherein the second gRNAmolecule positions a cleavage event 3′ to the target position (relativeto the target position on the first strand) on the second strand. In oneembodiment, the first gRNA molecule positions a cleavage event 3′ to thetarget position on the first strand.

In one embodiment, the second gRNA molecule positions a cleavage event5′ to the target position (relative to the target position on the firststrand) on the second strand. In one embodiment, the first gRNA moleculepositions a cleavage event 3′ to the target position on the firststrand, and wherein the second gRNA molecule positions a cleavage event5′ to the target position (relative to the target position on the firststrand) on the second strand. In one embodiment, the first gRNA moleculepositions a cleavage event 5′ to the target position on the firststrand, and wherein the second gRNA molecule positions a cleavage event5′ to the target position (relative to the target position on the firststrand) on the second strand, e.g., to produce a 5′ overhang. In oneembodiment, the first gRNA molecule positions a cleavage event 3′ to thetarget position on the first strand, and wherein the second gRNAmolecule positions a cleavage event 3′ to the target position (relativeto the target position on the first strand) on the second strand, e.g.,to produce a 5′ overhang. In one embodiment, the first gRNA moleculepositions a cleavage event 5′ to the target position on the firststrand, and wherein the second gRNA molecule positions a cleavage event5′ to the target position (relative to the target position on the firststrand) on the second strand, e.g., to produce a 3′ overhang. In oneembodiment, the first gRNA molecule positions a cleavage event 3′ to thetarget position on the first strand, and wherein the second gRNAmolecule positions a cleavage event 3′ to the target position (relativeto the target position on the first strand) on the second strand, e.g.,to produce a 3′ overhang. In one embodiment, the target positioncomprises a mutation. In one embodiment, the mutation is associated witha disease phenotype.

In one embodiment, the first gRNA molecule positions a cleavage event ona strand that binds to the gRNA molecule.

In one embodiment, the second gRNA molecule positions a cleavage eventon a strand that binds to the second gRNA molecule.

In one embodiment, the first gRNA molecule positions a cleavage event ona strand that binds to the gRNA and the second gRNA molecule positions acleavage event on a strand that binds to the second gRNA molecule, andwherein the first gRNA molecule and the second gRNA molecule bind todifferent strands, e.g., resulting in a 5′ overhang on each strand.

In one embodiment, the gRNA molecule, together with the Cas9 molecule(e.g., a nickase), positions a cleavage event on a strand (e.g., a firststrand or a second strand),

In one embodiment, the gRNA molecule positions a cleavage event 5′ tothe target position on the first strand. This embodiment allows the useof a single Cas9 molecule, e.g., a single Cas9 molecule that is anickase (e.g., a Cas9 molecule with a D10A mutation), e.g., to place asingle-strand cleavage event sufficiently close to the target position(e.g., within 10000, 9000, 8000, 7000, 6000, 5000, 4000, 3000, 2000,1000, 800, 600, 500, 400, 300, 200, 100, 75, 50, 40, 30, 20, 10, 5, or 1bp to the target position).

In one embodiment, the gRNA molecule positions a cleavage event 3′ tothe target position (relative to the target position on the firststrand) on the second strand. This embodiment allows the use of a singleCas9 molecule, e.g., a single Cas9 molecule that is a nickase (e.g., aCas9 molecule with a D10A mutation), e.g., to place a single-strandcleavage event sufficiently close to the target position (e.g., within10000, 9000, 8000, 7000, 6000, 5000, 4000, 3000, 2000, 1000, 800, 600,500, 400, 300, 200, 100, 75, 50, 40, 30, 20, 10, 5, or 1 bp to thetarget position).

In one embodiment, the gRNA molecule positions a cleavage event 3′ tothe target position on the first strand. This embodiment allows the useof a single Cas9 molecule, e.g., a single Cas9 molecule that is anickase (e.g., a Cas9 molecule with a D10A mutation), e.g., to place asingle-strand cleavage event sufficiently close to the target position(e.g., within 10000, 9000, 8000, 7000, 6000, 5000, 4000, 3000, 2000,1000, 800, 600, 500, 400, 300, 200, 100, 75, 50, 40, 30, 20, 10, 5, or 1bp to the target position).

In one embodiment, the gRNA molecule positions a cleavage event 5′ tothe target position (relative to the target position on the firststrand) on the second strand. This embodiment allows the use of a singleCas9 molecule, e.g., a single Cas9 molecule that is a nickase (e.g., aCas9 molecule with a D10A mutation), e.g., to place a single-strandcleavage event sufficiently close to the target position (e.g., within10000, 9000, 8000, 7000, 6000, 5000, 4000, 3000, 2000, 1000, 800, 600,500, 400, 300, 200, 100, 75, 50, 40, 30, 20, 10, 5, or 1 bp to thetarget position).

In one embodiment, the gRNA molecule positions a cleavage event 5′ tothe target position on the first strand. This embodiment allows the useof a single Cas9 molecule, e.g., a single Cas9 molecule that is anickase (e.g., a Cas9 molecule with an N863A mutation), e.g., to place asingle-strand cleavage event sufficiently close to the target position(e.g., within 10000, 9000, 8000, 7000, 6000, 5000, 4000, 3000, 2000,1000, 800, 600, 500, 400, 300, 200, 100, 75, 50, 40, 30, 20, 10, 5, or 1bp to the target position).

In one embodiment, the gRNA molecule positions a cleavage event 3′ tothe target position (relative to the target position on the firststrand) on the second strand. This embodiment allows the use of a singleCas9 molecule, e.g., a single Cas9 molecule that is a nickase (e.g., aCas9 molecule with an N863A mutation), e.g., to place a single-strandcleavage event sufficiently close to the target position (e.g., within10000, 9000, 8000, 7000, 6000, 5000, 4000, 3000, 2000, 1000, 800, 600,500, 400, 300, 200, 100, 75, 50, 40, 30, 20, 10, 5, or 1 bp to thetarget position).

In one embodiment, the gRNA molecule positions a cleavage event 3′ tothe target position on the first strand. This embodiment allows the useof a single Cas9 molecule, e.g., a single Cas9 molecule that is anickase (e.g., a Cas9 molecule with an N863A mutation), e.g., to place asingle-strand cleavage event sufficiently close to the target position(e.g., within 10000, 9000, 8000, 7000, 6000, 5000, 4000, 3000, 2000,1000, 800, 600, 500, 400, 300, 200, 100, 75, 50, 40, 30, 20, 10, 5, or 1bp to the target position).

In one embodiment, the gRNA molecule positions a cleavage event 5′ tothe target position (relative to the target position on the firststrand) on the second strand. This embodiment allows the use of a singleCas9 molecule, e.g., a single Cas9 molecule that is a nickase (e.g., aCas9 molecule with an N863A mutation), e.g., to place a single-strandcleavage event sufficiently close to the target position (e.g., within10000, 9000, 8000, 7000, 6000, 5000, 4000, 3000, 2000, 1000, 800, 600,500, 400, 300, 200, 100, 75, 50, 40, 30, 20, 10, 5, or 1 bp to thetarget position).

In one embodiment the gRNA molecule, together with the Cas9 molecule(e.g., a nickase), positions a cleavage event on the strand that bindsto the gRNA molecule; and the second gRNA molecule, together with theCas9 molecule, positions a cleavage event on the strand that binds tothe second gRNA molecule, wherein the gRNA molecule and the second gRNAmolecule bind to different strands, the gRNA molecule positions acleavage event 5′ to the target position on the first strand, and thesecond gRNA molecule positions a cleavage event 3′ to the targetposition (relative to the target position on the first strand) on thesecond strand. This embodiment allows the use of a single Cas9 molecule,e.g., a single Cas9 molecule that is a nickase (e.g., a Cas9 moleculewith a D10A mutation), e.g., to place single-strand cleavage events oneach side of the target position.

In one embodiment, the cleavage event positioned by the gRNA moleculeand the cleavage event positioned by the second gRNA molecule areseparated by 10 to 10000, 10 to 5000, 10 to 2500, 10 to 1000, 10 to 750,10 to 500, 10 to 400, 10 to 300, 10 to 200, 10 to 100, 10 to 75, 10 to50, or 10 to 25 base pairs.

In one embodiment:

the gRNA molecule, together with the Cas9 molecule (a nickase),positions a cleavage event on the strand other than the strand thatbinds to the gRNA molecule; and

the second gRNA molecule, together with the Cas9 molecule, positions acleavage event on the strand other than the strand that binds to thesecond gRNA molecule,

wherein:

the gRNA molecule and the second gRNA molecule bind to differentstrands,

the gRNA molecule positions a cleavage event 5′ to the target positionon the first strand, and

the second gRNA molecule positions a cleavage event 3′ to the targetposition (relative to the target position on the first strand) on thesecond strand. This embodiment allows the use of a single Cas9 molecule,e.g., a single Cas9 molecule that is a nickase (e.g., a Cas9 moleculewith an N863A mutation), e.g., to place single-strand cleavage events oneach side of the target position.

In one embodiment, the cleavage event positioned by the gRNA moleculeand the cleavage event positioned by the second gRNA molecule areseparated by 10 to 10000, 10 to 5000, 10 to 2500, 10 to 1000, 10 to 750,10 to 500, 10 to 400, 10 to 300, 10 to 200, 10 to 100, 10 to 75, 10 to50, or 10 to 25 base pairs.

In one embodiment:

the gRNA molecule, together with the Cas9 molecule (a nickase),positions a cleavage event on the strand that binds to the gRNAmolecule; and

the second gRNA molecule, together with the Cas9 molecule, positions acleavage event on the strand that binds to the second gRNA molecule,

wherein:

the gRNA molecule and the second gRNA molecule bind to differentstrands,

the gRNA molecule positions a cleavage event 3′ to the target positionon the first strand, and

the second gRNA molecule positions a cleavage event 5′ to the targetposition (relative to the target position on the first strand) on thesecond strand. This embodiment allows the use of a single Cas9 molecule,e.g., a single Cas9 molecule that is a nickase (e.g., a Cas9 moleculewith a D10A mutation), e.g., to place single-strand cleavage events oneach side of the target position.

In one embodiment, the cleavage event positioned by the gRNA moleculeand the cleavage event positioned by the second gRNA molecule areseparated by 10 to 10000, 10 to 5000, 10 to 2500, 10 to 1000, 10 to 750,10 to 500, 10 to 400, 10 to 300, 10 to 200, 10 to 100, 10 to 75, 10 to50, or 10 to 25 base pairs.

In one embodiment:

the gRNA molecule, together with the Cas9 molecule (a nickase),positions a cleavage event on the strand other than the strand thatbinds to the gRNA molecule; and

the second gRNA molecule, together with the Cas9 molecule, positions acleavage event on the strand other than the strand that binds to thesecond gRNA molecule,

wherein:

the gRNA molecule and the second gRNA molecule bind to differentstrands,

the gRNA molecule positions a cleavage event 3′ to the target positionon the first strand, and

the second gRNA molecule positions a cleavage event 5′ to the targetposition (relative to the target position on the first strand) on thesecond strand. This embodiment allows the use of a single Cas9 molecule,e.g., a single Cas9 molecule that is a nickase (e.g., a Cas9 moleculewith a N863A mutation), e.g., to place single-strand cleavage events oneach side of the target position.

In one embodiment, the cleavage event positioned by the gRNA moleculeand the cleavage event positioned by the second gRNA molecule areseparated by 10 to 10000, 10 to 5000, 10 to 2500, 10 to 1000, 10 to 750,10 to 500, 10 to 400, 10 to 300, 10 to 200, 10 to 100, 10 to 75, 10 to50, or 10 to 25 base pairs.

In one embodiment:

the gRNA molecule, together with the Cas9 molecule (e.g., a nickase),positions a cleavage event on the strand that binds to the gRNAmolecule; and

the second gRNA molecule, together with the Cas9 molecule, positions acleavage event on the strand that binds to the second gRNA molecule,

wherein:

the gRNA molecule and the second gRNA molecule bind to differentstrands,

the gRNA molecule positions a cleavage event 5′ to the target positionon the first strand, and

the second gRNA molecule positions a cleavage event 5′ to the targetposition (relative to the target position on the first strand) on thesecond strand, e.g., to produce a 5′ overhang. This embodiment allowsthe use of a single Cas9 molecule, e.g., a single Cas9 molecule that isa nickase (e.g., a Cas9 molecule with a D10A mutation), e.g., to placesingle-strand cleavage events on one side of the target position, e.g.,to produce a 5′ overhang.

In one embodiment, the cleavage event positioned by the gRNA moleculeand the cleavage event positioned by the second gRNA molecule areseparated by 10 to 10000, 10 to 5000, 10 to 2500, 10 to 1000, 10 to 750,10 to 500, 10 to 400, 10 to 300, 10 to 200, 10 to 100, 10 to 75, 10 to50, or 10 to 25 base pairs.

In one embodiment:

the gRNA molecule, together with the Cas9 molecule (a nickase),positions a cleavage event on the strand other than the strand thatbinds to the gRNA molecule; and

the second gRNA molecule, together with the Cas9 molecule, positions acleavage event on the strand other than the strand that binds to thesecond gRNA molecule,

wherein:

the gRNA molecule and the second gRNA molecule bind to differentstrands,

the gRNA molecule positions a cleavage event 5′ to the target positionon the first strand, and

the second gRNA molecule positions a cleavage event 5′ to the targetposition (relative to the target position on the first strand) on thesecond strand, e.g., to produce a 5′ overhang. This embodiment allowsthe use of a single Cas9 molecule, e.g., a single Cas9 molecule that isa nickase (e.g., a Cas9 molecule with an N863A mutation), e.g., to placesingle-strand cleavage events on each side of the target position, e.g.,to produce a 5′ overhang.

In one embodiment, the cleavage event positioned by the gRNA moleculeand the cleavage event positioned by the second gRNA molecule areseparated by 10 to 10000, 10 to 5000, 10 to 2500, 10 to 1000, 10 to 750,10 to 500, 10 to 400, 10 to 300, 10 to 200, 10 to 100, 10 to 75, 10 to50, or 10 to 25 base pairs.

In one embodiment:

the gRNA molecule, together with the Cas9 molecule (e.g., a nickase),positions a cleavage event on the strand that binds to the gRNAmolecule; and

the second gRNA molecule, together with the Cas9 molecule, positions acleavage event on the strand that binds to the second gRNA molecule,

wherein:

the gRNA molecule and the second gRNA molecule bind to differentstrands,

the gRNA molecule positions a cleavage event 3′ to the target positionon the first strand, and

the second gRNA molecule positions a cleavage event 3′ to the targetposition (relative to the target position on the first strand) on thesecond strand, e.g., to produce a 5′ overhang. This embodiment allowsthe use of a single Cas9 molecule, e.g., a single Cas9 molecule that isa nickase (e.g., a Cas9 molecule with a D10A mutation), e.g., to placesingle-strand cleavage events on one side of the target position, e.g.,to produce a 5′ overhang.

In one embodiment, the cleavage event positioned by the gRNA moleculeand the cleavage event positioned by the second gRNA molecule areseparated by 10 to 10000, 10 to 5000, 10 to 2500, 10 to 1000, 10 to 750,10 to 500, 10 to 400, 10 to 300, 10 to 200, 10 to 100, 10 to 75, 10 to50, or 10 to 25 base pairs.

In one embodiment:

the gRNA molecule, together with the Cas9 molecule (a nickase),positions a cleavage event on the strand other than the strand thatbinds to the gRNA molecule; and

the second gRNA molecule, together with the Cas9 molecule, positions acleavage event on the strand other than the strand that binds to thesecond gRNA molecule,

wherein:

the gRNA molecule and the second gRNA molecule bind to differentstrands,

the gRNA molecule positions a cleavage event 3′ to the target positionon the first strand, and

the second gRNA molecule positions a cleavage event 3′ to the targetposition (relative to the target position on the first strand) on thesecond strand, e.g., to produce a 5′ overhang. This embodiment allowsthe use of a single Cas9 molecule, e.g., a single Cas9 molecule that isa nickase (e.g., a Cas9 molecule with an N863A mutation), e.g., to placesingle-strand cleavage events on each side of the target position, e.g.,to produce a 5′ overhang.

In one embodiment, the cleavage event positioned by the gRNA moleculeand the cleavage event positioned by the second gRNA molecule areseparated by 10 to 10000, 10 to 5000, 10 to 2500, 10 to 1000, 10 to 750,10 to 500, 10 to 400, 10 to 300, 10 to 200, 10 to 100, 10 to 75, 10 to50, or 10 to 25 base pairs.

In one embodiment:

the gRNA molecule, together with the Cas9 molecule (e.g., a nickase),positions a cleavage event on the strand that binds to the gRNAmolecule; and

the second gRNA molecule, together with the Cas9 molecule, positions acleavage event on the strand that binds to the second gRNA molecule,

wherein:

the gRNA molecule and the second gRNA molecule bind to differentstrands,

the gRNA molecule positions a cleavage event 5′ to the target positionon the first strand, and

the second gRNA molecule positions a cleavage event 5′ to the targetposition (relative to the target position on the first strand) on thesecond strand, e.g., to produce a 3′ overhang. This embodiment allowsthe use of a single Cas9 molecule, e.g., a single Cas9 molecule that isa nickase (e.g., a Cas9 molecule with a D10A mutation), e.g., to placesingle-strand cleavage events on one side of the target position, e.g.,to produce a 3′ overhang.

In one embodiment, the cleavage event positioned by the gRNA moleculeand the cleavage event positioned by the second gRNA molecule areseparated by 10 to 10000, 10 to 5000, 10 to 2500, 10 to 1000, 10 to 750,10 to 500, 10 to 400, 10 to 300, 10 to 200, 10 to 100, 10 to 75, 10 to50, or 10 to 25 base pairs.

In one embodiment:

the gRNA molecule, together with the Cas9 molecule (a nickase),positions a cleavage event on the strand other than the strand thatbinds to the gRNA molecule; and

the second gRNA molecule, together with the Cas9 molecule, positions acleavage event on the strand other than the strand that binds to thesecond gRNA molecule,

wherein:

the gRNA molecule and the second gRNA molecule bind to differentstrands,

the gRNA molecule positions a cleavage event 5′ to the target positionon the first strand, and

the second gRNA molecule positions a cleavage event 5′ to the targetposition (relative to the target position on the first strand) on thesecond strand, e.g., to produce a 3′ overhang. This embodiment allowsthe use of a single Cas9 molecule, e.g., a single Cas9 molecule that isa nickase (e.g., a Cas9 molecule with an N863A mutation), e.g., to placesingle-strand cleavage events on each side of the target position, e.g.,to produce a 3′ overhang.

In one embodiment, the cleavage event positioned by the gRNA moleculeand the cleavage event positioned by the second gRNA molecule areseparated by 10 to 10000, 10 to 5000, 10 to 2500, 10 to 1000, 10 to 750,10 to 500, 10 to 400, 10 to 300, 10 to 200, 10 to 100, 10 to 75, 10 to50, or 10 to 25 base pairs.

In one embodiment:

the gRNA molecule, together with the Cas9 molecule (e.g., a nickase),positions a cleavage event on the strand that binds to the gRNAmolecule; and

the second gRNA molecule, together with the Cas9 molecule, positions acleavage event on the strand that binds to the second gRNA molecule,

wherein:

the gRNA molecule and the second gRNA molecule bind to differentstrands,

the gRNA molecule positions a cleavage event 3′ to the target positionon the first strand, and

the second gRNA molecule positions a cleavage event 3′ to the targetposition (relative to the target position on the first strand) on thesecond strand, e.g., to produce a 3′ overhang. This embodiment allowsthe use of a single Cas9 molecule, e.g., a single Cas9 molecule that isa nickase (e.g., a Cas9 molecule with a D10A mutation), e.g., to placesingle-strand cleavage events on one side of the target position, e.g.,to produce a 3′ overhang.

In one embodiment, the cleavage event positioned by the gRNA moleculeand the cleavage event positioned by the second gRNA molecule areseparated by 10 to 10000, 10 to 5000, 10 to 2500, 10 to 1000, 10 to 750,10 to 500, 10 to 400, 10 to 300, 10 to 200, 10 to 100, 10 to 75, 10 to50, or 10 to 25 base pairs.

In one embodiment:

the gRNA molecule, together with the Cas9 molecule (a nickase),positions a cleavage event on the strand other than the strand thatbinds to the gRNA molecule; and

the second gRNA molecule, together with the Cas9 molecule, positions acleavage event on the strand other than the strand that binds to thesecond gRNA molecule,

wherein:

the gRNA molecule and the second gRNA molecule bind to differentstrands,

the gRNA molecule positions a cleavage event 3′ to the target positionon the first strand, and

the second gRNA molecule positions a cleavage event 3′ to the targetposition (relative to the target position on the first strand) on thesecond strand, e.g., to produce a 3′ overhang. This embodiment allowsthe use of a single Cas9 molecule, e.g., a single Cas9 molecule that isa nickase (e.g., a Cas9 molecule with an N863A mutation), e.g., to placesingle-strand cleavage events on each side of the target position, e.g.,to produce a 3′ overhang.

In one embodiment, the cleavage event positioned by the gRNA moleculeand the cleavage event positioned by the second gRNA molecule areseparated by 10 to 10000, 10 to 5000, 10 to 2500, 10 to 1000, 10 to 750,10 to 500, 10 to 400, 10 to 300, 10 to 200, 10 to 100, 10 to 75, 10 to50, or 10 to 25 base pairs.

Homology Arms of the Donor Template

A homology arm should extend at least as far as the region in which endresection may occur, e.g., in order to allow the resected singlestranded overhang to find a complementary region within the donortemplate. The overall length could be limited by parameters such asplasmid size or viral packaging limits. In one embodiment, a homologyarm does not extend into repeated elements, e.g., Alu repeats or LINErepeats.

Exemplary homology arm lengths include at least 50, 100, 250, 500, 750,1000, 2000, 3000, 4000, or 5000 nucleotides. In some embodiments, thehomology arm length is 50-100, 100-250, 250-500, 500-750, 750-1000,1000-2000, 2000-3000, 3000-4000, or 4000-5000 nucleotides.

Target position, as used herein, refers to a site on a target nucleicacid (e.g., the chromosome) that is modified by a Cas9molecule-dependent process. For example, the target position can be amodified Cas9 molecule cleavage of the target nucleic acid and templatenucleic acid directed modification, e.g., correction, of the targetposition. In one embodiment, a target position can be a site between twonucleotides, e.g., adjacent nucleotides, on the target nucleic acid intowhich one or more nucleotides is added. The target position may compriseone or more nucleotides that are altered, e.g., corrected, by a templatenucleic acid. In one embodiment, the target position is within a targetsequence (e.g., the sequence to which the gRNA binds). In oneembodiment, a target position is upstream or downstream of a targetsequence (e.g., the sequence to which the gRNA binds).

A template nucleic acid, as that term is used herein, refers to anucleic acid sequence which can be used in conjunction with a Cas9molecule and a gRNA molecule to alter the structure of a targetposition. In certain embodiments, the target nucleic acid is modified tohave the some or all of the sequence of the template nucleic acid,typically at or near cleavage site(s). In one embodiment, the templatenucleic acid is single stranded. In certain embodiments, the templatenucleic acid is double stranded. In certain embodiments, the templatenucleic acid is DNA, e.g., double stranded DNA. In other embodiments,the template nucleic acid is single stranded DNA. In certainembodiments, the template nucleic acid is encoded on the same vectorbackbone, e.g., AAV genome, plasmid DNA, as the Cas9 and gRNA. In oneembodiment, the template nucleic acid is excised from a vector backbonein vivo, e.g., it is flanked by gRNA recognition sequences. In certainembodiments, the template nucleic acid comprises endogenous genomicsequence. In certain embodiments, a template nucleic acid is a templatenucleic acid covalently linked to the Cas9 molecule. In certainembodiments, a template nucleic acid is a template nucleic acidnon-covalently linked to the Cas9 molecule.

In certain embodiments, the template nucleic acid alters the structureof the target position by participating in an HDR event. In certainembodiments, the template nucleic acid alters the sequence of the targetposition. In certain embodiments, the template nucleic acid results inthe incorporation of a modified, or non-naturally occurring base intothe target nucleic acid.

Typically, the template sequence undergoes a breakage mediated orcatalyzed recombination with the target sequence. In certainembodiments, the template nucleic acid includes sequence thatcorresponds to a site on the target sequence that is cleaved by aneaCas9 mediated cleavage event. In certain embodiments, the templatenucleic acid includes sequence that corresponds to both, a first site onthe target sequence that is cleaved in a first Cas9 mediated event, anda second site on the target sequence that is cleaved in a second Cas9mediated event.

In one embodiment, the template nucleic acid can include sequence whichresults in an alteration in the coding sequence of a translatedsequence, e.g., one which results in the substitution of one amino acidfor another in a protein product, e.g., transforming a mutant alleleinto a wild type allele, transforming a wild type allele into a mutantallele, and/or introducing a stop codon, insertion of an amino acidresidue, deletion of an amino acid residue, or a nonsense mutation.

In other embodiments, the template nucleic acid can include sequencewhich results in an alteration in a non-coding sequence, e.g., analteration in an exon or in a 5′ or 3′ non-translated or non-transcribedregion. Such alterations include an alteration in a control element,e.g., a promoter, enhancer, and an alteration in a cis-acting ortrans-acting control element.

A template nucleic acid having homology with a target position in thetarget gene can be used to alter the structure of a target sequence(e.g., to correct a mutation present in a target position of anendogenous target gene). The template sequence can be used to alter anunwanted structure, e.g., an unwanted or mutant nucleotide.

A template nucleic acid typically comprises the following components:

[5′ homology arm]-[replacement sequence]-[3′ homology arm].

The homology arms provide for recombination into the chromosome, thusreplacing the undesired element, e.g., a mutation or signature, with thereplacement sequence. In certain embodiments, the homology arms flankthe most distal cleavage sites.

In certain embodiments, the 3′ end of the 5′ homology arm is theposition next to the 5′ end of the replacement sequence. In oneembodiment, the 5′ homology arm can extend at least 10, 20, 30, 40, 50,100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 3000,4000, or 5000 nucleotides 5′ from the 5′ end of the replacementsequence.

In certain embodiments, the 5′ end of the 3′ homology arm is theposition next to the 3′ end of the replacement sequence. In certainembodiments, the 3′ homology arm can extend at least 10, 20, 30, 40, 50,100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 3000,4000, or 5000 nucleotides 3′ from the 3′ end of the replacementsequence.

In certain embodiments, to alter one or more nucleotides at a targetposition (e.g., to correct a mutation), the homology arms, e.g., the 5′and 3′ homology arms, may each comprise about 1000 bp of sequenceflanking the most distal gRNAs (e.g., 1000 bp of sequence on either sideof the target position (e.g., the mutation).

It is contemplated herein that one or both homology arms may beshortened to avoid including certain sequence repeat elements, e.g., Alurepeats or LINE elements. For example, a 5′ homology arm may beshortened to avoid a sequence repeat element. In other embodiments, a 3′homology arm may be shortened to avoid a sequence repeat element. Insome embodiments, both the 5′ and the 3′ homology arms may be shortenedto avoid including certain sequence repeat elements.

It is contemplated herein that template nucleic acids for altering thesequence (e.g., correcting a mutation) of a target position may bedesigned for use as a single-stranded oligonucleotide, e.g., asingle-stranded oligodeoxynucleotide (ssODN). When using a ssODN, 5′ and3′ homology arms may range up to about 200 bp in length, e.g., at least25, 50, 75, 100, 125, 150, 175, or 200 bp in length. Longer homologyarms are also contemplated for ssODNs as improvements in oligonucleotidesynthesis continue to be made. In some embodiments, a longer homologyarm is made by a method other than chemical synthesis, e.g., bydenaturing a long double stranded nucleic acid and purifying one of thestrands, e.g., by affinity for a strand-specific sequence anchored to asolid substrate.

While not wishing to be bound by theory, in certain embodiments alt-HDRproceeds more efficiently when the template nucleic acid has extendedhomology 5′ to the nick (i.e., in the 5′ direction of the nickedstrand). Accordingly, in some embodiments, the template nucleic acid hasa longer homology arm and a shorter homology arm, wherein the longerhomology arm can anneal 5′ of the nick. In some embodiments, the armthat can anneal 5′ to the nick is at least 25, 50, 75, 100, 125, 150,175, or 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 3000,4000, or 5000 nucleotides from the nick or the 5′ or 3′ end of thereplacement sequence. In some embodiments, the arm that can anneal 5′ tothe nick is at least 10%, 20%, 30%, 40%, or 50% longer than the arm thatcan anneal 3′ to the nick. In some embodiments, the arm that can anneal5′ to the nick is at least 2×, 3×, 4×, or 5× longer than the arm thatcan anneal 3′ to the nick. Depending on whether a ssDNA template cananneal to the intact strand or the nicked strand, the homology arm thatanneals 5′ to the nick may be at the 5′ end of the ssDNA template or the3′ end of the ssDNA template, respectively.

Similarly, in some embodiments, the template nucleic acid has a 5′homology arm, a replacement sequence, and a 3′ homology arm, such thatthe template nucleic acid has extended homology to the 5′ of the nick.For example, the 5′ homology arm and 3′ homology arm may besubstantially the same length, but the replacement sequence may extendfarther 5′ of the nick than 3′ of the nick. In some embodiments, thereplacement sequence extends at least 10%, 20%, 30%, 40%, 50%, 2×, 3×,4×, or 5× further to the 5′ end of the nick than the 3′ end of the nick.

While not wishing to be bound by theory, In some embodiments, alt-HDRproceeds more efficiently when the template nucleic acid is centered onthe nick. Accordingly, in some embodiments, the template nucleic acidhas two homology arms that are essentially the same size. For instance,the first homology arm of a template nucleic acid may have a length thatis within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the secondhomology arm of the template nucleic acid.

Similarly, in some embodiments, the template nucleic acid has a 5′homology arm, a replacement sequence, and a 3′ homology arm, such thatthe template nucleic acid extends substantially the same distance oneither side of the nick. For example, the homology arms may havedifferent lengths, but the replacement sequence may be selected tocompensate for this. For example, the replacement sequence may extendfurther 5′ from the nick than it does 3′ of the nick, but the homologyarm 5′ of the nick is shorter than the homology arm 3′ of the nick, tocompensate. The converse is also possible, e.g., that the replacementsequence may extend further 3′ from the nick than it does 5′ of thenick, but the homology arm 3′ of the nick is shorter than the homologyarm 5′ of the nick, to compensate.

Exemplary Template Nucleic Acids

In a preferred embodiment, and in order to increase DNA repair via genecorrection, the template nucleic acid is linked to the Cas9 molecule aspart of a Cas9 fusion molecule. In certain embodiments, the templatenucleic acid is double stranded. In other embodiments, the templatenucleic acid is single stranded. In certain embodiments, the templatenucleic acid comprises a single stranded portion and a double strandedportion. In certain embodiments, the template nucleic acid comprisesabout 50 to 100, e.g., 55 to 95, 60 to 90, 65 to 85, or 70 to 80 bp,homology on either side of the nick and/or replacement sequence. Incertain embodiments, the template nucleic acid comprises about 50, 55,60, 65, 70, 75, 80, 85, 90, 95, or 100 bp homology 5′ of the nick orreplacement sequence, 3′ of the nick or replacement sequence, or both 5′and 3′ of the nick or replacement sequences.

In certain embodiments, the template nucleic acid comprises about 150 to200 bp, e.g., 155 to 195, 160 to 190, 165 to 185, or 170 to 180 bp,homology 3′ of the nick and/or replacement sequence. In certainembodiments, the template nucleic acid comprises about 150, 155, 160,165, 170, 175, 180, 185, 190, 195, or 200 bp homology 3′ of the nick orreplacement sequence. In some embodiments, the template nucleic acidcomprises less than about 100, 90, 80, 70, 60, 50, 40, 30, 20, 15, or 10bp homology 5′ of the nick or replacement sequence.

In certain embodiments, the template nucleic acid comprises about 150 to200 bp, e.g., 155 to 195, 160 to 190, 165 to 185, or 170 to 180 bp,homology 5′ of the nick and/or replacement sequence. In certainembodiments, the template nucleic acid comprises about 150, 155, 160,165, 170, 175, 180, 185, 190, 195, or 200 bp homology 5′ of the nick orreplacement sequence. In certain embodiments, the template nucleic acidcomprises less than about 100, 90, 80, 70, 60, 50, 40, 30, 20, 15, or 10bp homology 3′ of the nick or replacement sequence.

In certain embodiments, the template nucleic acid comprises a nucleotidesequence, e.g., of one or more nucleotides, that will be added to orwill template a change in the target nucleic acid. In other embodiments,the template nucleic acid comprises a nucleotide sequence that may beused to modify the target position. In other embodiments, the templatenucleic acid comprises a nucleotide sequence, e.g., of one or morenucleotides, that corresponds to wild type sequence of the targetnucleic acid, e.g., of the target position.

The template nucleic acid may comprise a replacement sequence. In someembodiments, the template nucleic acid comprises a 5′ homology arm. Insome embodiments, the template nucleic acid comprises a 3′ homology arm.

In certain embodiments, the template nucleic acid is linear doublestranded DNA. The length may be, e.g., about 150-200 bp, e.g., about150, 160, 170, 180, 190, or 200 bp. The length may be, e.g., at least150, 160, 170, 180, 190, or 200 bp. In some embodiments, the length isno greater than 150, 160, 170, 180, 190, or 200 bp. In some embodiments,a double stranded template nucleic acid has a length of about 160 bp,e.g., about 155-165, 150-170, 140-180, 130-190, 120-200, 110-210,100-220, 90-230, or 80-240 bp.

The template nucleic acid can be linear single stranded DNA. In certainembodiments, the template nucleic acid is (i) linear single stranded DNAthat can anneal to the nicked strand of the target nucleic acid, (ii)linear single stranded DNA that can anneal to the intact strand of thetarget nucleic acid, (iii) linear single stranded DNA that can anneal tothe plus strand of the target nucleic acid, (iv) linear single strandedDNA that can anneal to the minus strand of the target nucleic acid, ormore than one of the preceding. The length may be, e.g., about 150-200nucleotides, e.g., about 150, 160, 170, 180, 190, or 200 nucleotides.The length may be, e.g., at least 150, 160, 170, 180, 190, or 200nucleotides. In some embodiments, the length is no greater than 150,160, 170, 180, 190, or 200 nucleotides. In some embodiments, a singlestranded template nucleic acid has a length of about 160 nucleotides,e.g., about 155-165, 150-170, 140-180, 130-190, 120-200, 110-210,100-220, 90-230, or 80-240 nucleotides.

In some embodiments, the template nucleic acid is circular doublestranded DNA, e.g., a plasmid. In some embodiments, the template nucleicacid comprises about 500 to 1000 bp of homology on either side of thereplacement sequence and/or the nick. In some embodiments, the templatenucleic acid comprises about 300, 400, 500, 600, 700, 800, 900, 1000,1500, or 2000 bp of homology 5′ of the nick or replacement sequence, 3′of the nick or replacement sequence, or both 5′ and 3′ of the nick orreplacement sequence. In some embodiments, the template nucleic acidcomprises at least 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or2000 bp of homology 5′ of the nick or replacement sequence, 3′ of thenick or replacement sequence, or both 5′ and 3′ of the nick orreplacement sequence. In some embodiments, the template nucleic acidcomprises no more than 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or2000 bp of homology 5′ of the nick or replacement sequence, 3′ of thenick or replacement sequence, or both 5′ and 3′ of the nick orreplacement sequence.

In certain embodiments, one or both homology arms may be shortened toavoid including certain sequence repeat elements, e.g., Alu repeats,LINE elements. For example, a 5′ homology arm may be shortened to avoida sequence repeat element, while a 3′ homology arm may be shortened toavoid a sequence repeat element. In some embodiments, both the 5′ andthe 3′ homology arms may be shortened to avoid including certainsequence repeat elements.

In some embodiments, the Cas9 fusion molecule, comprising the templatenucleic acid, is in an adenovirus vector, e.g., an AAV vector, e.g., assDNA molecule of a length and sequence that allows it to be packaged inan AAV capsid. The vector may be, e.g., less than 5 kb and may containan ITR sequence that promotes packaging into the capsid. The vector maybe integration-deficient. In some embodiments, the template nucleic acidcomprises about 150 to 1000 nucleotides of homology on either side ofthe replacement sequence and/or the nick. In some embodiments, thetemplate nucleic acid comprises about 100, 150, 200, 300, 400, 500, 600,700, 800, 900, 1000, 1500, or 2000 nucleotides 5′ of the nick orreplacement sequence, 3′ of the nick or replacement sequence, or both 5′and 3′ of the nick or replacement sequence. In some embodiments, thetemplate nucleic acid comprises at least 100, 150, 200, 300, 400, 500,600, 700, 800, 900, 1000, 1500, or 2000 nucleotides 5′ of the nick orreplacement sequence, 3′ of the nick or replacement sequence, or both 5′and 3′ of the nick or replacement sequence. In some embodiments, thetemplate nucleic acid comprises at most 100, 150, 200, 300, 400, 500,600, 700, 800, 900, 1000, 1500, or 2000 nucleotides 5′ of the nick orreplacement sequence, 3′ of the nick or replacement sequence, or both 5′and 3′ of the nick or replacement sequence.

In some embodiments, the Cas9 fusion molecule, comprising the templatenucleic acid, is in a lentiviral vector, e.g., an DLV (integrationdeficiency lentivirus). In some embodiments, the template nucleic acidcomprises about 500 to 1000 base pairs of homology on either side of thereplacement sequence and/or the nick. In some embodiments, the templatenucleic acid comprises about 300, 400, 500, 600, 700, 800, 900, 1000,1500, or 2000 bp of homology 5′ of the nick or replacement sequence, 3′of the nick or replacement sequence, or both 5′ and 3′ of the nick orreplacement sequence. In some embodiments, the template nucleic acidcomprises at least 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or2000 bp of homology 5′ of the nick or replacement sequence, 3′ of thenick or replacement sequence, or both 5′ and 3′ of the nick orreplacement sequence. In some embodiments, the template nucleic acidcomprises no more than 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or2000 bp of homology 5′ of the nick or replacement sequence, 3′ of thenick or replacement sequence, or both 5′ and 3′ of the nick orreplacement sequence.

In certain embodiments, the template nucleic acid alters the structureof the target position by participating in an HDR event. In someembodiments, the template nucleic acid alters the sequence of the targetposition. In some embodiments, the template nucleic acid results in theincorporation of a modified, or non-naturally occurring nucleotide baseinto the target nucleic acid.

Typically, the template sequence undergoes a breakage mediated orcatalyzed recombination with the target sequence. In some embodiments,the template nucleic acid includes sequence that corresponds to a siteon the target sequence that is cleaved by an eaCas9 mediated cleavageevent. In some embodiments, the template nucleic acid includes sequencethat corresponds to both, a first site on the target sequence that iscleaved in a first Cas9 mediated event, and a second site on the targetsequence that is cleaved in a second Cas9 mediated event.

In some embodiments, the template nucleic acid can include sequencewhich results in an alteration in the coding sequence of a translatedsequence, e.g., one which results in the substitution of one amino acidfor another in a protein product, e.g., transforming a mutant alleleinto a wild type allele, transforming a wild type allele into a mutantallele, and/or introduction of a stop codon, insertion of an amino acidresidue, deletion of an amino acid residue, or a nonsense mutation.

In some embodiments, the template nucleic acid can include sequencewhich results in an alteration in a non-coding sequence, e.g., analteration in an exon or in a 5′ or 3′ non-translated or non-transcribedregion. Such alterations include an alteration in a control element,e.g., a promoter or enhancer, or an alteration in a cis-acting ortrans-acting control element.

In some embodiments, a template nucleic acid having homology with atarget position can be used to alter the structure of a target sequence.The template nucleic acid sequence can be used to alter an unwantedstructure, e.g., an unwanted or mutant nucleotide.

In some embodiments, shorter homology arms, e.g., 5′ and/or 3′ homologyarms may be used. In certain embodiments, the length of the 5′ homologyarm is about 5 to about 100 nucleotides. In some embodiments, the lengthof the 5′ homology arm is about 10 to about 150 nucleotides. In someembodiments, the length of the 5′ homology arm is about 20 to about 150nucleotides. In certain embodiments, the length of the 5′ homology armis about 10, 20, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550,600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, or morenucleotides in length.

In certain embodiments, the length of the 3′ homology arm is about 5 toabout 100 nucleotides. In some embodiments, the length of the 3′homology arm is about 10 to about 150 nucleotides. In some embodiments,the length of the 3′ homology arm is about 20 to about 150 nucleotides.In certain embodiments, the length of the 3′ homology arm is about 10,20, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700,750, 800, 850, 900, 950, 1000, 1100, 1200, or more nucleotides inlength.

It is contemplated herein that one or both homology arms may beshortened to avoid including certain sequence repeat elements, e.g., Alurepeats, LINE elements. For example, a 5′ homology arm may be shortenedto avoid a sequence repeat element. In one embodiment, a 3′ homology armmay be shortened to avoid a sequence repeat element. In one embodiment,both the 5′ and the 3′ homology arms may be shortened to avoid includingcertain sequence repeat elements. In some embodiments, the length of the5′ homology arm is at least 50 nucleotides in length, but not longenough to include a repeated element. In some embodiments, the length ofthe 5′ homology arm is at least 100 nucleotides in length, but not longenough to include a repeated element. In some embodiments, the length ofthe 5′ homology arm is at least 150 nucleotides in length, but not longenough to include a repeated element. In some embodiments, the length ofthe 3′ homology arm is at least 50 nucleotides in length, but not longenough to include a repeated element. In some embodiments, the length ofthe 3′ homology arm is at least 100 nucleotides in length, but not longenough to include a repeated element. In some embodiments, the length ofthe 3′ homology arm is at least 150 nucleotides in length, but not longenough to include a repeated element.

It is contemplated herein that template nucleic acids for correcting amutation may be designed for use as a single-stranded oligonucleotide(ssODN), e.g., a single-stranded oligodeoxynucleotide. When using assODN, 5′ and 3′ homology arms may range up to about 200 bp in length,e.g., at least 25, 50, 75, 100, 125, 150, 175, or 200 bp in length.Longer homology arms are also contemplated for ssODNs as improvements inoligonucleotide synthesis continue to be made.

Silent Mutations in the Template Nucleic Acid

It is contemplated herein that Cas9 could potentially cleave donorconstructs either prior to or following homology directed repair (e.g.,homologous recombination), resulting in a possiblenon-homologous-end-joining event and further DNA sequence mutation atthe chromosomal locus of interest. Therefore, to avoid cleavage of thedonor sequence before and/or after Cas9-mediated homology directedrepair, in some embodiments, alternate versions of the donor sequencemay be used where silent mutations are introduced. These silentmutations may disrupt Cas9 binding and cleavage, but not disrupt theamino acid sequence of the repaired gene. For example, mutations mayinclude those made to a donor sequence to repair the target gene, themutant form of which can cause disease.

Increasing Gene Correction

In certain embodiments of the methods provided herein, the frequency ofpreferred repair outcomes generated using a Cas9 fusion moleculedescribed herein may be increased as compared to the frequency ofpreferred repair outcomes with a Cas9 fusion molecule and a templatenucleic acid which are not fused. In some embodiments, the frequency ofgene correction resulting from a Cas9 fusion molecule induced-lesion ina target position of a target cell overexpressing a gene correctionpathway component is increased at least about 1-fold, at least about2-fold, at least about 3-fold, at least about 4-fold, at least about5-fold, at least about 6-fold, at least about 7-fold, at least about8-fold, at least about 9-fold, at least about 10-fold, or more, ascompared to the frequency of gene correction resulting from a Cas9molecule and a target nucleic acid which are not fused in a targetposition.

In some embodiments, the frequency of gene correction resulting from aCas9 fusion molecule induced-lesion in a target position of a targetcell overexpressing a gene correction pathway component is increased atleast 5% (e.g., at least about 5%, at least about 10%, at least about15%, at least about 20%, at least about 25%, at least about 30%, atleast about 35%, at least about 40%, at least about 45%, at least about50%, at least about 55%, at least about 60%, at least about 65%, atleast about 70%, at least about 75%, at least about 80%, at least about85%, at least about 90%, at least about 95%, at least about 100%, atleast about 150%, at least about 200%, at least about 300%, at leastabout 400%, at least about 500%, at least about 600%, at least about700%, at least about 800%, at least about 900%, or more.

NHEJ Approaches for Gene Targeting

In certain embodiments of the methods provided herein, NHEJ-mediateddeletion is used to delete all or part of a target gene. As describedherein, nuclease-induced NHEJ can also be used to remove (e.g., delete)sequences in a gene of interest.

While not wishing to be bound by theory, it is believed that, in certainembodiments, the genomic alterations associated with the methodsdescribed herein rely on nuclease-induced NHEJ and the error-pronenature of the NHEJ repair pathway. NHEJ repairs a double-strand break inthe DNA by joining together the two ends; however, generally, theoriginal sequence is restored only if two compatible ends, exactly asthey were formed by the double-strand break, are perfectly ligated. TheDNA ends of the double-strand break are frequently the subject ofenzymatic processing, resulting in the addition or removal ofnucleotides, e.g., resection, at one or both strands, prior to rejoiningof the ends. This results in the presence of insertion and/or deletion(indel) mutations in the DNA sequence at the site of the NHEJ repair.Two-thirds of these mutations typically alter the reading frame and,therefore, produce a non-functional protein. Additionally, mutationsthat maintain the reading frame, but which insert or delete asignificant amount of sequence, can destroy functionality of theprotein. This is locus dependent as mutations in critical functionaldomains are likely less tolerable than mutations in non-critical regionsof the protein.

The indel mutations generated by NHEJ are unpredictable in nature;however, at a given break site certain indel sequences are favored andare over represented in the population, likely due to small regions ofmicrohomology. The lengths of deletions can vary widely; most commonlyin the 1-50 bp range, but they can easily reach greater than 100-200 bp.In some embodiments, the deletion is at least about 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 47, 50, 75,100, 200, 300, 400, 500, 750, 1000, 2000, 3000, 4000, 5000, 6000, 7000,8000, 9000, 10000, 15000, 20000, 25000, 30000, 40000, 50000, 60000,70000, 80000, 90000, 100000, 200000, 300000, 400000, 500000, 600000,700000, 800000, 900000, 1000000 or more nucleotides in length.Insertions tend to be shorter and often include short duplications ofthe sequence immediately surrounding the break site. However, it ispossible to obtain large insertions, and in these cases, the insertedsequence has often been traced to other regions of the genome or toplasmid DNA present in the cells.

Because NHEJ is a mutagenic process, it can also be used to delete smallsequence motifs as long as the generation of a specific final sequenceis not required. If a double-strand break is targeted near to a shorttarget sequence, the deletion mutations caused by the NHEJ repair oftenspan, and therefore remove, the unwanted nucleotides. For the deletionof larger DNA segments, introducing two double-strand breaks, one oneach side of the sequence, can result in NHEJ between the ends withremoval of the entire intervening sequence. Both of these approaches canbe used to delete specific DNA sequences; however, the error-pronenature of NHEJ may still produce indel mutations at the site of repair.

Both double-strand cleaving eaCas9 molecules and single strand, ornickase, eaCas9 molecules can be used in the methods and compositionsdescribed herein to generate NHEJ-mediated indels. NHEJ-mediated indelstargeted to the gene, e.g., a coding region, e.g., an early codingregion of a gene of interest can be used to knockout (i.e., eliminateexpression of) a gene of interest. For example, early coding region of agene of interest includes sequence immediately following a transcriptionstart site, within a first exon of the coding sequence, or within 500 bpof the transcription start site (e.g., less than 500, 450, 400, 350,300, 250, 200, 150, 100 or 50 bp).

Placement of Double-Strand or Single-Strand Breaks Relative to theTarget Position

In certain embodiments, in which a gRNA and Cas9 nuclease generate adouble-strand break for the purpose of inducing NHEJ-mediated indels, agRNA, e.g., a unimolecular (or chimeric) or modular gRNA molecule, isconfigured to position one double-strand break in close proximity to anucleotide of the target position. In one embodiment, the cleavage siteis between 0-30 bp away from the target position (e.g., less than 30,25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 bp from the targetposition).

In certain embodiments, in which two gRNAs complexing with Cas9 nickasesinduce two single-strand breaks for the purpose of inducingNHEJ-mediated indels, two gRNAs, e.g., independently, unimolecular (orchimeric) or modular gRNA, are configured to position two single-strandbreaks to provide for NHEJ repair a nucleotide of the target position.In certain embodiments, the gRNAs are configured to position cuts at thesame position, or within a few nucleotides of one another, on differentstrands, essentially mimicking a double-strand break. In certainembodiments, the closer nick is between 0-30 bp away from the targetposition (e.g., less than 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or1 bp from the target position), and the two nicks are within 25-55 bp ofeach other (e.g., between 25 to 50, 25 to 45, 25 to 40, 25 to 35, 25 to30, 50 to 55, 45 to 55, 40 to 55, 35 to 55, 30 to 55, 30 to 50, 35 to50, 40 to 50, 45 to 50, 35 to 45, or 40 to 45 bp) and no more than 100bp away from each other (e.g., no more than 90, 80, 70, 60, 50, 40, 30,20, or 10 bp). In certain embodiments, the gRNAs are configured to placea single-strand break on either side of a nucleotide of the targetposition.

Both double-strand cleaving eaCas9 molecules and single strand, ornickase, eaCas9 molecules can be used in the methods and compositionsdescribed herein to generate breaks both sides of a target position.Double-strand or paired single-strand breaks may be generated on bothsides of a target position to remove the nucleic acid sequence betweenthe two cuts (e.g., the region between the two breaks in deleted). Incertain embodiments, two gRNAs, e.g., independently, unimolecular (orchimeric) or modular gRNA, are configured to position a double-strandbreak on both sides of a target position. In other embodiments, threegRNAs, e.g., independently, unimolecular (or chimeric) or modular gRNA,are configured to position a double-strand break (i.e., one gRNAcomplexes with a Cas9 nuclease) and two single-strand breaks or pairedsingle-strand breaks (i.e., two gRNAs complex with Cas9 nickases) oneither side of the target position. In certain embodiments, four gRNAs,e.g., independently, unimolecular (or chimeric) or modular gRNA, areconfigured to generate two pairs of single-strand breaks (i.e., twopairs of two gRNAs complex with Cas9 nickases) on either side of thetarget position. The double-strand break(s) or the closer of the twosingle-strand nicks in a pair will ideally be within 0-500 bp of thetarget position (e.g., no more than 450, 400, 350, 300, 250, 200, 150,100, 50, or 25 bp from the target position). When nickases are used, thetwo nicks in a pair are within 25-55 bp of each other (e.g., between 25to 50, 25 to 45, 25 to 40, 25 to 35, 25 to 30, 50 to 55, 45 to 55, 40 to55, 35 to 55, 30 to 55, 30 to 50, 35 to 50, 40 to 50, 45 to 50, 35 to45, or 40 to 45 bp) and no more than 100 bp away from each other (e.g.,no more than 90, 80, 70, 60, 50, 40, 30, 20, or 10 bp).

Targeted Knockdown

Unlike CRISPR/Cas-mediated gene knockout, which permanently eliminatesexpression by mutating the gene at the DNA level, CRISPR/Cas knockdownallows for temporary reduction of gene expression through the use ofartificial transcription factors. Mutating key residues in both DNAcleavage domains of the Cas9 molecule (e.g., the D10A and H840Amutations) results in the generation of a catalytically inactive Cas9(referred to herein as “eiCas9”, which is also known as dead Cas9 ordCas9) molecule. An eiCas9 complexes with a gRNA and localizes to theDNA sequence specified by that gRNA's targeting domain, however, it doesnot cleave the target DNA. Fusion of the eiCas9 to an effector domain,e.g., a transcription repression domain, enables recruitment of theeffector to any DNA site specified by the gRNA. Although an eiCas9itself can block transcription when recruited to early regions in thecoding sequence, more robust repression can be achieved by fusing atranscriptional repression domain (for example KRAB, SID or ERD) to theeiCas9, referred to herein as a “Cas9-repressor”, and recruiting thetranscriptional repression domain to the target knockdown position,e.g., within 1000 bp of sequence 3′ of the start codon or within 500 bpof a promoter region 5′ of the start codon of a gene. It is likely thattargeting DNAse I hypersensitive sites (DHSs) of the promoter may yieldmore efficient gene repression or activation because these regions aremore likely to be accessible to the eiCas9 and are also more likely toharbor sites for endogenous transcription factors. Especially for generepression, it is contemplated herein that blocking the binding site ofan endogenous transcription factor would aid in downregulating geneexpression. In certain embodiments, one or more eiCas9 molecules may beused to block binding of one or more endogenous transcription factors.In some embodiments, an eiCas9 molecule can be fused to a chromatinmodifying protein. Altering chromatin status can result in decreasedexpression of the target gene. One or more eiCas9 molecules fused to oneor more chromatin modifying proteins may be used to alter chromatinstatus.

In one embodiment, a gRNA molecule can be targeted to a knowntranscription response elements (e.g., promoters, enhancers, etc.), aknown upstream activating sequences (UAS), and/or sequences of unknownor known function that are suspected of being able to control expressionof the target DNA.

CRISPR/Cas-mediated gene knockdown can be used to reduce expression ofan unwanted allele or transcript. Contemplated herein are scenarioswherein permanent destruction of the gene is not ideal. In thesescenarios, site-specific repression may be used to temporarily reduce oreliminate expression. It is also contemplated herein that the off-targeteffects of a Cas9-repressor may be less severe than those of aCas9-nuclease as a nuclease can cleave any DNA sequence and causemutations whereas a Cas9-repressor may only have an effect if it targetsthe promoter region of an actively transcribed gene. However, whilenuclease-mediated knockout is permanent, repression may only persist aslong as the Cas9-repressor is present in the cells. Once the repressoris no longer present, it is likely that endogenous transcription factorsand gene regulatory elements would restore expression to its naturalstate.

Single-Strand Annealing

Single-strand annealing (SSA) is another DNA repair process that repairsa double-strand break between two repeat sequences present in a targetnucleic acid. Repeat sequences utilized by the SSA pathway are generallygreater than 30 nucleotides in length. Resection at the break endsoccurs to reveal repeat sequences on both strands of the target nucleicacid. After resection, single-strand overhangs containing the repeatsequences are coated with RPA protein to prevent the repeats sequencesfrom inappropriate annealing, e.g., to themselves. RAD52 binds to andeach of the repeat sequences on the overhangs and aligns the sequencesto enable the annealing of the complementary repeat sequences. Afterannealing, the single-strand flaps of the overhangs are cleaved. New DNAsynthesis fills in any gaps, and ligation restores the DNA duplex. As aresult of the processing, the DNA sequence between the two repeats isdeleted. The length of the deletion can depend on many factors includingthe location of the two repeats utilized, and the pathway orprocessivity of the resection.

In contrast to HDR pathways, SSA does not require a template nucleicacid to alter or correct a target nucleic acid sequence. Instead, thecomplementary repeat sequence is utilized.

Other DNA Repair Pathways

SSBR (Single-Strand Break Repair)

Single-stranded breaks (SSB) in the genome are repaired by the SSBRpathway, which is a distinct mechanism from the DSB repair mechanismsdiscussed above. The SSBR pathway has four major stages: SSB detection,DNA end processing, DNA gap filling, and DNA ligation. A more detailedexplanation is given in Caldecott 2008, and a summary is given here.

In the first stage, when a SSB forms, PARP1 and/or PARP2 recognize thebreak and recruit repair machinery. The binding and activity of PARP1 atDNA breaks is transient and it seems to accelerate SSBr by promoting thefocal accumulation or stability of SSBr protein complexes at the lesion.Arguably the most important of these SSBr proteins is XRCC1, whichfunctions as a molecular scaffold that interacts with, stabilizes, andstimulates multiple enzymatic components of the SSBr process includingthe protein responsible for cleaning the DNA 3′ and 5′ ends. Forinstance, XRCC1 interacts with several proteins (DNA polymerase beta,PNK, and three nucleases, APE1, APTX, and APLF) that promote endprocessing. APE1 has endonuclease activity. APLF exhibits endonucleaseand 3′ to 5′ exonuclease activities. APTX has endonuclease and 3′ to 5′exonuclease activity.

This end processing is an important stage of SSBR since the 3′- and/or5′-termini of most, if not all, SSBs are damaged. End processinggenerally involves restoring a damaged 3′-end to a hydroxylated stateand and/or a damaged 5′ end to a phosphate moiety, so that the endsbecome ligation-competent. Enzymes that can process damaged 3′ terminiinclude PNKP, APE1, and TDP1. Enzymes that can process damaged 5′termini include PNKP, DNA polymerase beta, and APTX. LIG3 (DNA ligaseIII) can also participate in end processing. Once the ends are cleaned,gap filling can occur.

At the DNA gap filling stage, the proteins typically present are PARP1,DNA polymerase beta, XRCC1, FEN1 (flap endonuclease 1), DNA polymerasedelta/epsilon, PCNA, and LIG1. There are two ways of gap filling, theshort patch repair and the long patch repair. Short patch repairinvolves the insertion of a single nucleotide that is missing. At someSSBs, “gap filling” might continue displacing two or more nucleotides(displacement of up to 12 bases have been reported). FEN1 is anendonuclease that removes the displaced 5′-residues. Multiple DNApolymerases, including Polβ, are involved in the repair of SSBs, withthe choice of DNA polymerase influenced by the source and type of SSB.

In the fourth stage, a DNA ligase such as LIG1 (Ligase I) or LIG3(Ligase III) catalyzes joining of the ends. Short patch repair usesLigase III and long patch repair uses Ligase I.

Sometimes, SSBR is replication-coupled. This pathway can involve one ormore of CtIP, MRN, ERCC1, and FEN1. Additional factors that may promoteSSBR include: aPARP, PARP1, PARP2, PARG, XRCC1, DNA polymerase (3, DNApolymerase delta, DNA polymerase epsilon, PCNA, LIG1, PNK, PNKP, APE1,APTX, APLF, TDP1, LIG3, FEN1, CtIP, MRN, and ERCC1.

MMR (Mismatch Repair)

Cells contain three excision repair pathways: MMR, BER, and NER. Theexcision repair pathways have a common feature in that they typicallyrecognize a lesion on one strand of the DNA, then exo/endonucleasesremove the lesion and leave a 1-30 nucleotide gap that issub-sequentially filled in by DNA polymerase and finally sealed withligase. A more complete picture is given in Li 2008, and a summary isprovided here.

Mismatch repair (MMR) operates on mispaired DNA bases.

The MSH2/6 or MSH2/3 complexes both have ATPase activity that plays animportant role in mismatch recognition and the initiation of repair.MSH2/6 preferentially recognizes base-base mismatches and identifiesmispairs of 1 or 2 nucleotides, while MSH2/3 preferentially recognizeslarger ID mispairs.

hMLH1 heterodimerizes with hPMS2 to form hMutLα which possesses anATPase activity and is important for multiple steps of MMR. It possessesa PCNA/replication factor C (RFC)-dependent endonuclease activity whichplays an important role in 3′ nick-directed MMR involving EXO1 (EXO1 isa participant in both HR and MMR). It regulates termination ofmismatch-provoked excision. Ligase I is the relevant ligase for thispathway. Additional factors that may promote MMR include: EXO1, MSH2,MSH3, MSH6, MLH1, PMS2, MLH3, DNA Pol delta, RPA, HMGB1, RFC, and DNAligase I.

Base Excision Repair (BER)

The base excision repair (BER) pathway is active throughout the cellcycle; it is responsible primarily for removing small,non-helix-distorting base lesions from the genome. In contrast, therelated Nucleotide Excision Repair pathway (discussed in the nextsection) repairs bulky helix-distorting lesions. A more detailedexplanation is given in Caldecott 2008, and a summary is given here.

Upon DNA base damage, base excision repair (BER) is initiated and theprocess can be simplified into five major steps: (a) removal of thedamaged DNA base; (b) incision of the subsequent a basic site; (c)clean-up of the DNA ends; (d) insertion of the desired nucleotide intothe repair gap; and (e) ligation of the remaining nick in the DNAbackbone. These last steps are similar to the SSBR.

In the first step, a damage-specific DNA glycosylase excises the damagedbase through cleavage of the N-glycosidic bond linking the base to thesugar phosphate backbone. Then AP endonuclease-1 (APE1) or bifunctionalDNA glycosylases with an associated lyase activity incises thephosphodiester backbone to create a DNA single-strand break (SSB). Thethird step of BER involves cleaning-up of the DNA ends. The fourth stepin BER is conducted by Pol β that adds a new complementary nucleotideinto the repair gap and in the final step XRCC1/Ligase III seals theremaining nick in the DNA backbone. This completes the short-patch BERpathway in which the majority (˜80%) of damaged DNA bases are repaired.However, if the 5′-ends in step 3 are resistant to end processingactivity, following one nucleotide insertion by Pol β there is then apolymerase switch to the replicative DNA polymerases, Pol δ/ε, whichthen add ˜2-8 more nucleotides into the DNA repair gap. This creates a5′-flap structure, which is recognized and excised by flapendonuclease-1 (FEN-1) in association with the processivity factorproliferating cell nuclear antigen (PCNA). DNA ligase I then seals theremaining nick in the DNA backbone and completes long-patch BER.Additional factors that may promote the BER pathway include: DNAglycosylase, APE1, Polβ, Pol delta, Pol epsilon, XRCC1, Ligase III,FEN-1, PCNA, RECQL4, WRN, MYH, PNKP, and APTX.

Nucleotide Excision Repair (NER)

Nucleotide excision repair (NER) is an important excision mechanism thatremoves bulky helix-distorting lesions from DNA. Additional detailsabout NER are given in Marteijn et al. 2014, and a summary is givenhere. NER a broad pathway encompassing two smaller pathways: globalgenomic NER (GG-NER) and transcription coupled repair NER (TC-NER).GG-NER and TC-NER use different factors for recognizing DNA damage.However, they utilize the same machinery for lesion incision, repair,and ligation.

Once damage is recognized, the cell removes a short single-stranded DNAsegment that contains the lesion. Endonucleases XPF/ERCC1 and XPG(encoded by ERCC5) remove the lesion by cutting the damaged strand oneither side of the lesion, resulting in a single-strand gap of 22-30nucleotides. Next, the cell performs DNA gap filling synthesis andligation. Involved in this process are: PCNA, RFC, DNA Pol δ, DNA Pol εor DNA Pol κ and DNA ligase I or XRCC1/Ligase III. Replicating cellstend to use DNA pol ε and DNA ligase I, while non-replicating cells tendto use DNA Pol δ, DNA Pol κ, and the XRCC1/Ligase III complex to performthe ligation step.

NER can involve the following factors: XPA-G, POLH, XPF, ERCC1, XPA-G,and LIG1. Transcription-coupled NER (TC-NER) can involve the followingfactors: CSA, CSB, XPB, XPD, XPG, ERCC1, and TTDA. Additional factorsthat may promote the NER repair pathway include XPA-G, POLH, XPF, ERCC1,XPA-G, LIG1, CSA, CSB, XPA, XPB, XPC, XPD, XPF, XPG, TTDA, UVSSA, USP7,CETN2, RAD23B, UV-DDB, CAK subcomplex, RPA, and PCNA.

Interstrand Crosslink (ICL)

A dedicated pathway called the ICL repair pathway repairs interstrandcrosslinks. Interstrand crosslinks, or covalent crosslinks between basesin different DNA strand, can occur during replication or transcription.ICL repair involves the coordination of multiple repair processes, inparticular, nucleolytic activity, translesion synthesis (TLS), and HDR.Nucleases are recruited to excise the ICL on either side of thecrosslinked bases, while TLS and HDR are coordinated to repair the cutstrands. ICL repair can involve the following factors: endonucleases,e.g., XPF and RAD51C, endonucleases such as RAD51, translesionpolymerases, e.g., DNA polymerase zeta and Rev1, and the Fanconi anemia(FA) proteins, e.g., FancJ.

Other Pathways

Several other DNA repair pathways exist in mammals.

Translesion synthesis (TLS) is a pathway for repairing a single strandedbreak left after a defective replication event and involves translesionpolymerases, e.g., DNA pol and Rev1.

Error-free postreplication repair (PRR) is another pathway for repairinga single stranded break left after a defective replication event.

Examples of gRNAs in Genome Editing Methods

gRNA molecules as described herein can be used with Cas9 molecules, orCas9 fusion molecules, that generate a double-strand break or asingle-strand break to alter the sequence of a target nucleic acid,e.g., a target position or target genetic signature. gRNA moleculesuseful in these methods are described below.

In certain embodiments, the gRNA, e.g., a chimeric gRNA, is configuredsuch that it comprises one or more of the following properties;

a) it can position, e.g., when targeting a Cas9 molecule, or Cas9 fusionmolecule, that makes double-strand breaks, a double-strand break (i)within 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 nucleotides ofa target position, or (ii) sufficiently close that the target positionis within the region of end resection;

b) it has a targeting domain of at least 16 nucleotides, e.g., atargeting domain of (i) 16, (ii), 17, (iii) 18, (iv) 19, (v) 20, (vi)21, (vii) 22, (viii) 23, (ix) 24, (x) 25, or (xi) 26 nucleotides; and

(c)(i) the proximal and tail domain, when taken together, comprise atleast 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides,e.g., at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53nucleotides from a naturally occurring S. pyogenes or S. aureus, tailand proximal domain, or a sequence that differs by no more than 1, 2, 3,4, 5, 6, 7, 8, 9, or 10 nucleotides therefrom;

(c)(ii) there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50,or 53 nucleotides 3′ to the last nucleotide of the secondcomplementarity domain, e.g., at least 15, 18, 20, 25, 30, 31, 35, 40,45, 49, 50, or 53 nucleotides from the corresponding sequence of anaturally occurring S. pyogenes or S. aureus gRNA, or a sequence thatdiffers by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotidestherefrom;

(c)(iii) there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51,or 54 nucleotides 3′ to the last nucleotide of the secondcomplementarity domain that is complementary to its correspondingnucleotide of the first complementarity domain, e.g., at least 16, 19,21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides from thecorresponding sequence of a naturally occurring S. pyogenes or S. aureusgRNA, or a sequence that differs by no more than 1, 2, 3, 4, 5, 6, 7, 8,9, or 10 nucleotides therefrom;

(c)(iv) the tail domain is at least 10, 15, 20, 25, 30, 35 or 40nucleotides in length, e.g., it comprises at least 10, 15, 20, 25, 30,35 or 40 nucleotides from a naturally occurring S. pyogenes or S. aureustail domain, or a sequence that differs by no more than 1, 2, 3, 4, 5,6, 7, 8, 9, or 10 nucleotides therefrom; or

(c)(v) the tail domain comprises 15, 20, 25, 30, 35, 40 nucleotides orall of the corresponding portions of a naturally occurring tail domain,e.g., a naturally occurring S. pyogenes or S. aureus tail domain.

In certain embodiments, the gRNA is configured such that it comprisesproperties a and b(i); a and b(ii); a and b(iii); a and b(iv); a andb(v); a and b(vi); a and b(vii); a and b(viii); a and b(ix); a and b(x);a and b(xi); a and c; a, b, and c; a(i), b(i), and c(i); a(i), b(i), andc(ii); a(i), b(ii), and c(i); a(i), b(ii), and c(ii); a(i), b(iii), andc(i); a(i), b(iii), and c(ii); a(i), b(iv), and c(i); a(i), b(iv), andc(ii); a(i), b(v), and c(i); a(i), b(v), and c(ii); a(i), b(vi), andc(i); a(i), b(vi), and c(ii); a(i), b(vii), and c(i); a(i), b(vii), andc(ii); a(i), b(viii), and c(i); a(i), b(viii), and c(ii); a(i), b(ix),and c(i); a(i), b(ix), and c(ii); a(i), b(x), and c(i); a(i), b(x), andc(ii); a(i), b(xi), or c(i); a(i), b(xi), and c(ii).

In certain embodiments, the gRNA, e.g., a chimeric gRNA, is configuredsuch that it comprises one or more of the following properties:

(a) one or both of the gRNAs can position, e.g., when targeting a Cas9molecule, or Cas9 fusion molecule, that makes single-strand breaks, asingle-strand break within (i) 50, 100, 150, 200, 250, 300, 350, 400,450, or 500 nucleotides of a target position, or (ii) sufficiently closethat the target position is within the region of end resection;

(b) one or both have a targeting domain of at least 16 nucleotides,e.g., a targeting domain of (i) 16, (ii), 17, (iii) 18, (iv) 19, (v) 20,(vi) 21, (vii) 22, (viii) 23, (ix) 24, (x) 25, or (xi) 26 nucleotides;and

(c)(i) the proximal and tail domain, when taken together, comprise atleast 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides,e.g., at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53nucleotides from a naturally occurring S. pyogenes or S. aureus tail andproximal domain, or a sequence that differs by no more than 1, 2, 3, 4,5, 6, 7, 8, 9, or 10 nucleotides therefrom;

(c)(ii) there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50,or 53 nucleotides 3′ to the last nucleotide of the secondcomplementarity domain, e.g., at least 15, 18, 20, 25, 30, 31, 35, 40,45, 49, 50, or 53 nucleotides from the corresponding sequence of anaturally occurring S. pyogenes, or S. aureus gRNA, or a sequence thatdiffers by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotidestherefrom;

(c)(iii) there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51,or 54 nucleotides 3′ to the last nucleotide of the secondcomplementarity domain that is complementary to its correspondingnucleotide of the first complementarity domain, e.g., at least 16, 19,21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides from thecorresponding sequence of a naturally occurring S. pyogenes or S. aureusgRNA, or a sequence that differs by no more than 1, 2, 3, 4, 5, 6, 7, 8,9, or 10 nucleotides therefrom;

(c)(iv) the tail domain is at least 10, 15, 20, 25, 30, 35 or 40nucleotides in length, e.g., it comprises at least 10, 15, 20, 25, 30,35 or 40 nucleotides from a naturally occurring S. pyogenes, or S.aureus tail domain, or a sequence that differs by no more than 1, 2, 3,4, 5, 6, 7, 8, 9, or 10 nucleotides therefrom; or

(c)(v) the tail domain comprises 15, 20, 25, 30, 35, 40 nucleotides orall of the corresponding portions of a naturally occurring tail domain,e.g., a naturally occurring S. pyogenes or S. aureus tail domain.

In certain embodiments, the gRNA is configured such that it comprisesproperties: a and b(i); a and b(ii); a and b(iii); a and b(iv); a andb(v); a and b(vi); a and b(vii); a and b(viii); a and b(ix); a and b(x);a and b(xi); a and c; a, b, and c; a(i), b(i), and c(i); a(i), b(i), andc(ii); a(i), b(ii), and c(i); a(i), b(ii), and c(ii); a(i), b(iii), andc(i); a(i), b(iii), and c(ii); a(i), b(iv), and c(i); a(i), b(iv), andc(ii); a(i), b(v), and c(i); a(i), b(v), and c(ii); a(i), b(vi), andc(i); a(i), b(vi), and c(ii); a(i), b(vii), and c(i); a(i), b(vii), andc(ii); a(i), b(viii), and c(i); a(i), b(viii), and c(ii); a(i), b(ix),and c(i); a(i), b(ix), and c(ii); a(i), b(x), and c(i); a(i), b(x), andc(ii); a(i), b(xi), and c(i); or a(i), b(xi), and c(ii).

In certain embodiments, the gRNA is used with a Cas9 nickase moleculehaving HNH activity, e.g., a Cas9 molecule, or a Cas9 fusion molecule,having the RuvC activity inactivated, e.g., a Cas9 molecule having amutation at D10, e.g., the D10A mutation.

In one embodiment, the gRNA is used with a Cas9 nickase molecule havingRuvC activity, e.g., a Cas9 molecule, or a Cas9 fusion molecule, havingthe HNH activity inactivated, e.g., a Cas9 molecule having a mutation at840, e.g., the H840A.

In one embodiment, the gRNAs are used with a Cas9 nickase moleculehaving RuvC activity, e.g., a Cas9 molecule, or a Cas9 fusion molecule,having the HNH activity inactivated, e.g., a Cas9 molecule having amutation at N863, e.g., the N863A mutation.

In embodiment, a pair of gRNAs, e.g., a pair of chimeric gRNAs,comprising a first and a second gRNA, is configured such that theycomprises one or more of the following properties:

a) one or both of the gRNA molecules can position, e.g., when targetinga Cas9 molecule, or a Cas9 fusion molecule, that makes single-strandbreaks, a single-strand break within (i) 50, 100, 150, 200, 250, 300,350, 400, 450, or 500 nucleotides of a target position, or (ii)sufficiently close that the target position is within the region of endresection;

b) one or both have a targeting domain of at least 16 nucleotides, e.g.,a targeting domain of (i) 16, (ii), 17, (iii) 18, (iv) 19, (v) 20, (vi)21, (vii) 22, (viii) 23, (ix) 24, (x) 25, or (xi) 26 nucleotides;

(c)(i) the proximal and tail domain, when taken together, comprise atleast 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides,e.g., at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53nucleotides from a naturally occurring S. pyogenes or S. aureus tail andproximal domain, or a sequence that differs by no more than 1, 2, 3, 4,5, 6, 7, 8, 9, or 10 nucleotides therefrom;

(c)(ii) there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50,or 53 nucleotides 3′ to the last nucleotide of the secondcomplementarity domain, e.g., at least 15, 18, 20, 25, 30, 31, 35, 40,45, 49, 50, or 53 nucleotides from the corresponding sequence of anaturally occurring S. pyogenes or S. aureus gRNA, or a sequence thatdiffers by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotidestherefrom;

(c)(iii) there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51,or 54 nucleotides 3′ to the last nucleotide of the secondcomplementarity domain that is complementary to its correspondingnucleotide of the first complementarity domain, e.g., at least 16, 19,21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides from thecorresponding sequence of a naturally occurring S. pyogenes or S. aureusgRNA, or a sequence that differs by no more than 1, 2, 3, 4, 5, 6, 7, 8,9, or 10 nucleotides therefrom;

(c)(iv) the tail domain is at least 10, 15, 20, 25, 30, 35 or 40nucleotides in length, e.g., it comprises at least 10, 15, 20, 25, 30,35 or 40 nucleotides from a naturally occurring S. pyogenes or S. aureustail domain; or, or a sequence that differs by no more than 1, 2, 3, 4,5, 6, 7, 8, 9, or 10 nucleotides therefrom; or

(c)(v) the tail domain comprises 15, 20, 25, 30, 35, or 40 nucleotidesor all of the corresponding portions of a naturally occurring taildomain, e.g., a naturally occurring S. pyogenes or S. aureus taildomain;

(d) the gRNAs are configured such that, when hybridized to targetnucleic acid, they are separated by 0-50, 0-100, 0-200, at least 10, atleast 20, at least 30 or at least 50 nucleotides;

(e) the breaks made by the first gRNA and second gRNA are on differentstrands; and

(f) the PAMs are facing outwards.

In certain embodiments, one or both of the gRNAs is configured such thatit comprises properties a and b(i); a and b(ii); a and b(iii); a andb(iv); a and b(v); a and b(vi); a and b(vii); a and b(viii); a andb(ix); a and b(x); a and b(xi); a and c; a, b, and c; a(i), b(i), andc(i); a(i), b(i), and c(ii); a(i), b(i), c, and d; a(i), b(i), c, and e;a(i), b(i), c, d, and e; a(i), b(ii), and c(i); a(i), b(ii), and c(ii);a(i), b(ii), c, and d; a(i), b(ii), c, and e; a(i), b(ii), c, d, and e;a(i), b(iii), and c(i); a(i), b(iii), and c(ii); a(i), b(iii), c, and d;a(i), b(iii), c, and e; a(i), b(iii), c, d, and e; a(i), b(iv), andc(i); a(i), b(iv), and c(ii); a(i), b(iv), c, and d; a(i), b(iv), c, ande; a(i), b(iv), c, d, and e; a(i), b(v), and c(i); a(i), b(v), andc(ii); a(i), b(v), c, and d; a(i), b(v), c, and e; a(i), b(v), c, d, ande; a(i), b(vi), and c(i); a(i), b(vi), and c(ii); a(i), b(vi), c, and d;a(i), b(vi), c, and e; a(i), b(vi), c, d, and e; a(i), b(vii), and c(i);a(i), b(vii), and c(ii); a(i), b(vii), c, and d; a(i), b(vii), c, and e;a(i), b(vii), c, d, and e; a(i), b(viii), and c(i); a(i), b(viii), andc(ii); a(i), b(viii), c, and d; a(i), b(viii), c, and e; a(i), b(viii),c, d, and e; a(i), b(ix), and c(i); a(i), b(ix), and c(ii); a(i), b(ix),c, and d; a(i), b(ix), c, and e; a(i), b(ix), c, d, and e; a(i), b(x),and c(i); a(i), b(x), and c(ii); a(i), b(x), c, and d; a(i), b(x), c,and e; a(i), b(x), c, d, and e; a(i), b(xi), and c(i); a(i), b(xi), andc(ii); a(i), b(xi), c, and d; a(i), b(xi), c, and e; or a(i), b(xi), c,d, and e.

In certain embodiments, the gRNAs are used with a Cas9 nickase moleculehaving HNH activity, e.g., a Cas9 molecule, or a Cas9 fusion molecule,having the RuvC activity inactivated, e.g., a Cas9 molecule having amutation at D10, e.g., the D10A mutation.

In certain embodiments, the gRNAs are used with a Cas9 nickase moleculehaving RuvC activity, e.g., a Cas9 molecule, or a Cas9 fusion molecule,having the HNH activity inactivated, e.g., a Cas9 molecule having amutation at H840, e.g., the H840A mutation.

In certain embodiments, the gRNAs are used with a Cas9 nickase moleculehaving RuvC activity, e.g., a Cas9 molecule, or a Cas9 fusion molecule,having the HNH activity inactivated, e.g., a Cas9 molecule having amutation at N863, e.g., the N863A mutation.

VI. Target Cells

Cas9 fusion molecules and gRNA molecules, e.g., a Cas9 (fusion)molecule/gRNA molecule complex, can be used to manipulate a cell, e.g.,to edit a target nucleic acid, in a wide variety of cells. Additionaldetails on types of cells that can be manipulated may be found in thesection entitled “VIIA. TARGETS: CELLS” of PCT Application WO2015/048577, the entire contents of which are expressly incorporatedherein by reference.

In certain embodiments, a cell is manipulated by editing (e.g.,introducing a mutation in) a target gene as described herein. In oneembodiment, a cell, or a population of cells, is manipulated by editingone or more non-coding sequences, e.g., an alteration in an intron or ina 5′ or 3′ non-translated or non-transcribed region. In one embodiment,a cell, or a population of cells, is manipulated by editing the sequenceof a control element, e.g., a promoter, enhancer, or a cis-acting ortrans-acting control element. In one embodiment, a cell, or a populationof cells, is manipulated by editing one or more coding sequences, e.g.,an alteration in an exon. In some embodiments, a cell, or a populationof cells, is manipulated in vitro. In other embodiments, a cell, or apopulation of cells, is manipulated ex vivo. In some embodiments, acell, or a population of cells, is manipulated in vivo. In someembodiments, the expression of one or more target genes (e.g., one ormore target genes described herein) is modulated, e.g., in vivo. Inother embodiments, the expression of one or more target genes (e.g., oneor more target genes described herein) is modulated, e.g., ex vivo. Inother embodiments, the expression of one or more target genes (e.g., oneor more target genes described herein) is modulated, e.g., in vitro.

In one embodiment, a cell, or a population of cells, is manipulated byediting (e.g., inducing a mutation in) the target gene, e.g., asdescribed herein. In one embodiment, the expression of the target geneis modulated, e.g., in vivo. In another embodiment, the expression ofthe target gene is modulated, e.g., ex vivo.

The Cas9 (or Cas9 fusion molecule) and gRNA molecules described hereincan be delivered to a target cell. In certain embodiments, the targetcell is an erythroid cell, e.g., an erythroblast. In certainembodiments, erythroid cells are preferentially targeted, e.g., at leastabout 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the targeted cells areerythroid cells. For example, in the case of in vivo delivery, erythroidcells are preferentially targeted, and if cells are treated ex vivo andreturned to the subject, erythroid cells are preferentially modified. Incertain embodiments, the target cell is a circulating blood cell, e.g.,a reticulocyte, megakaryocyte erythroid progenitor (MEP) cell, myeloidprogenitor cell (CMP/GMP), lymphoid progenitor (LP) cell, hematopoieticstem/progenitor cell (HSC), or endothelial cell (EC). In certainembodiments, the target cell is a bone marrow cell (e.g., areticulocyte, an erythroid cell (e.g., erythroblast), an MEP cell,myeloid progenitor cell (CMP/GMP), LP cell, erythroid progenitor (EP)cell, HSC, multipotent progenitor (MPP) cell, endothelial cell (EC),hemogenic endothelial (HE) cell, or mesenchymal stem cell). In certainembodiments, the target cell is a myeloid progenitor cell (e.g., acommon myeloid progenitor (CMP) cell or granulocyte macrophageprogenitor (GMP) cell). In certain embodiments, the target cell is alymphoid progenitor cell, e.g., a common lymphoid progenitor (CLP) cell.In certain embodiments, the target cell is an erythroid progenitor cell(e.g., an MEP cell). In certain embodiments, the target cell is ahematopoietic stem/progenitor cell (e.g., a long term HSC (LT-HSC),short term HSC (ST-HSC), MPP cell, or lineage restricted progenitor(LRP) cell). In certain embodiments, the target cell is a CD34⁺ cell,CD34⁺CD90⁺ cell, CD34⁺CD38⁻ cell, CD34⁺CD90⁺CD49f⁺CD38⁻CD45RA⁻ cell,CD105⁺ cell, CD31⁺, or CD133⁺ cell, or a CD34⁺CD90⁺CD133⁺ cell. Incertain embodiments, the target cell is an umbilical cord blood CD34⁺HSPC, umbilical cord venous endothelial cell, umbilical cord arterialendothelial cell, amniotic fluid CD34⁺ cell, amniotic fluid endothelialcell, placental endothelial cell, or placental hematopoietic CD34⁺ cell.In certain embodiments, the target cell is a mobilized peripheral bloodhematopoietic CD34⁺ cell (after the patient is treated with amobilization agent, e.g., G-CSF or Plerixafor). In certain embodiments,the target cell is a peripheral blood endothelial cell.

In certain embodiments, a target cell is manipulated ex vivo by editing(e.g., inducing a mutation in) the target gene and/or modulating theexpression of the target gene, then the target cell is administered tothe subject. Sources of target cells for ex vivo manipulation mayinclude, for example, the subject's blood, cord blood, or marrow. Othersources of target cells for ex vivo manipulation may include, forexample, heterologous donor blood, cord blood, or bone marrow.

In certain embodiments, an erythrocyte is removed from a subject,manipulated ex vivo as described above, and the erythrocyte is returnedto the subject. In other embodiments, a hematopoietic stem cell isremoved from a subject, manipulated ex vivo as described above, and thehematopoietic stem cell is returned to the subject. In certainembodiments, an erythroid progenitor cell is removed from a subject,manipulated ex vivo as described above, and the erythroid progenitorcell is returned to the subject. In certain embodiments, an myeloidprogenitor cell is removed from a subject, manipulated ex vivo asdescribed above, and the myeloid progenitor cell is returned to thesubject. In certain embodiments, a hematopoietic stem/progenitor cell(HSC) is removed from a subject, manipulated ex vivo as described above,and returned to the subject. In certain embodiments, a CD34⁺ HSC isremoved from a subject, manipulated ex vivo as described above, andreturned to the subject.

In certain embodiments wherein modified HSCs generated ex vivo areadministered to a subject without myeloblative pre-conditioning. Inother embodiments, the modified HSCs are administered after mildmyeloblative conditioning such that, followed engraftment, some of thehematopoietic cells are derived from the modified HSCs. In still otherembodiments, the modified HSCs are administered after full myeloblationsuch that, following engraftment, 100% of the hematopoietic cells arederived from the modified HSCs.

A suitable cell can also include a stem cell such as, by way of example,an embryonic stem cell, induced pluripotent stem cell, hematopoieticstem cell, or hemogenic endothelial (HE) cell (precursor to bothhematopoietic stem cells and endothelial cells). In certain embodiments,the cell is an induced pluripotent stem (iPS) cell or a cell derivedfrom an iPS cell, e.g., an iPS cell generated from the subject, modifiedusing methods disclosed herein and differentiated into a clinicallyrelevant cell such as e.g., an erythrocyte. In one embodiment, AAV isused to transduce the target cells, e.g., the target cells describedherein.

Cells produced by the methods described herein may be used immediately.Alternatively, the cells may be frozen (e.g., in liquid nitrogen) andstored for later use. The cells will usually be frozen in 10%dimethylsulfoxide (DMSO), 50% serum, 40% buffered medium, or some othersuch solution as is commonly used in the art to preserve cells at suchfreezing temperature and thawed in such a manner as commonly known inthe art for thawing frozen cultured cells. Cells may also bethermostabilized for prolonged storage at 4° C.

Delivery, Formulations and Routes of Administration

The components, e.g., a Cas9 fusion molecule and at least one gRNAmolecule (e.g., a Cas9 fusion molecule/gRNA molecule complex), can bedelivered, formulated, or administered, in a variety of forms, see,e.g., Tables 5-6. In certain embodiments, the sequence(s) encoding thetwo or more (e.g., 2, 3, 4, or more) different gRNA molecules arepresent on the same nucleic acid molecule, e.g., an AAV vector. When agRNA component is delivered encoded in DNA, the DNA will typicallyinclude a control region, e.g., comprising a promoter, to effectexpression. In one embodiment, the promoter is a constitutive promoter.In another embodiment, the promoter is a tissue specific promoter.Useful promoters for gRNAs include T7, H1, EF-1a, U6, U1, and tRNApromoters. Promoters with similar or dissimilar strengths can beselected to tune the expression of components. Sequences encoding theCas9 molecule of a Cas9 fusion molecule can comprise a nuclearlocalization signal (NLS), e.g., an SV40 NLS. In one embodiment, thesequence encoding the Cas9 molecule of a Cas9 fusion molecule comprisesat least two nuclear localization signals. In one embodiment a promoterfor the Cas9 molecule of a Cas9 fusion molecule or a gRNA molecule canbe, independently, inducible, tissue specific, or cell specific.

Table 5 provides examples of how the components can be formulated,delivered, or administered.

TABLE 5 Elements Optional Donor Cas9 Template Fusion gRNA NucleicMolecule(s) Molecule(s) Acid Comments Protein DNA DNA In thisembodiment, a Cas9 fusion molecule, typically an eaCas9 fusion molecule,is provided as a protein, covalently or non-covalently lined to thetemplate nucleic acid, and a gRNA molecule is transcribed from DNA.Protein RNA DNA In this embodiment, a Cas9 fusion molecule, typically aneaCas9 fusion molecule, is provided as a protein, covalently ornon-covalently lined to the template nucleic acid, and a gRNA moleculeis provided as transcribed or synthesized RNA

Table 6 summarizes various delivery methods for the components of a Cassystem, e.g., the Cas9 molecule component and the gRNA moleculecomponent, as described herein.

TABLE 6 Delivery into Non- Type of Dividing Duration of Genome MoleculeDelivery Vector/Mode Cells Expression Integration Delivered Physical(e.g., YES Transient NO Nucleic electroporation, particle gun, Acids andcalcium phosphate Proteins transfection, cell compression or squeezing)Viral Retrovirus NO Stable YES RNA Lentivirus YES Stable YES/NO with RNAmodifications Adenovirus YES Transient NO DNA Adeno- YES Stable NO DNAAssociated Virus (AAV) Vaccinia Virus YES Very NO DNA Transient HerpesSimplex YES Stable NO DNA Virus Non-Viral Cationic YES Transient Dependson Nucleic Liposomes what is Acids and delivered Proteins Polymeric YESTransient Depends on Nucleic Nanoparticles what is Acids and deliveredProteins Biological Attenuated YES Transient NO Nucleic Non-ViralBacteria Acids Delivery Engineered YES Transient NO Nucleic VehiclesBacteriophages Acids Mammalian YES Transient NO Nucleic Virus-like AcidsParticles Biological YES Transient NO Nucleic liposomes: AcidsErythrocyte Ghosts and ExosomesDNA-Based Delivery of One or More gRNA Molecules

Nucleic acids encoding gRNA molecules can be administered to subjects ordelivered into cells by art-known methods or as described herein. Forexample, gRNA-encoding DNA can be delivered by, e.g., vectors (e.g.,viral or non-viral vectors), non-vector based methods (e.g., using nakedDNA or DNA complexes), or a combination thereof.

Nucleic acids encoding gRNA molecules can be conjugated to molecules(e.g., N-acetylgalactosamine) promoting uptake by the target cells(e.g., erythrocytes, HSCs).

In some embodiments, the gRNA-encoding DNA is delivered by a vector(e.g., viral vector/virus or plasmid).

Vectors can comprise a sequence that encodes a gRNA molecule. One ormore regulatory/control elements, e.g., promoters, enhancers, introns,polyadenylation signals, Kozak consensus sequences, internal ribosomeentry sites (IRES), can be included in the vectors. In some embodiments,the promoter is recognized by RNA polymerase II (e.g., a CMV promoter).In other embodiments, the promoter is recognized by RNA polymerase III(e.g., a U6 promoter). In some embodiments, the promoter is a regulatedpromoter (e.g., inducible promoter). In other embodiment, the promoteris a constitutive promoter. In some embodiments, the promoter is atissue specific promoter. In other embodiments, the promoter is a viralpromoter. In some embodiments, the promoter is a non-viral promoter.

In some embodiments, the vector is a viral vector (e.g., for generationof recombinant viruses). In some embodiments, the virus is a DNA virus(e.g., dsDNA or ssDNA virus). In other embodiments, the virus is an RNAvirus (e.g., an ssRNA virus). In some embodiments, the virus infectsdividing cells. In other embodiments, the virus infects non-dividingcells. Exemplary viral vectors/viruses include, e.g., retroviruses,lentiviruses, adenovirus, adeno-associated virus (AAV), vacciniaviruses, poxviruses, and herpes simplex viruses.

In some embodiments, the virus infects both dividing and non-dividingcells. In some embodiments, the virus can integrate into the hostgenome. In some embodiments, the virus is engineered to have reducedimmunity, e.g., in human. In some embodiments, the virus isreplication-competent. In other embodiments, the virus isreplication-defective, e.g., having one or more coding regions for thegenes necessary for additional rounds of virion replication and/orpackaging replaced with other genes or deleted. In some embodiments, thevirus causes transient expression of the gRNA molecule. In otherembodiments, the virus causes long-lasting, e.g., at least 1 week, 2weeks, 1 month, 2 months, 3 months, 6 months, 9 months, 1 year, 2 years,or permanent expression, of the gRNA molecule. The packaging capacity ofthe viruses may vary, e.g., from at least about 4 kb to at least about30 kb, e.g., at least about 5 kb, 10 kb, 15 kb, 20 kb, 25 kb, 30 kb, 35kb, 40 kb, 45 kb, or 50 kb.

In one embodiment, the viral vector recognizes a specific cell type ortissue. For example, the viral vector can be pseudotyped with adifferent/alternative viral envelope glycoprotein; engineered with acell type-specific receptor (e.g., genetic modification(s) of one ormore viral envelope glycoproteins to incorporate a targeting ligand suchas a peptide ligand, a single chain antibody, or a growth factor);and/or engineered to have a molecular bridge with dual specificitieswith one end recognizing a viral glycoprotein and the other endrecognizing a moiety of the target cell surface (e.g., aligand-receptor, monoclonal antibody, avidin-biotin and chemicalconjugation).

In some embodiments, the gRNA-encoding nucleic acid sequence isdelivered by a recombinant retrovirus. In some embodiments, theretrovirus (e.g., Moloney murine leukemia virus) comprises a reversetranscriptase, e.g., that allows integration into the host genome. Insome embodiments, the retrovirus is replication-competent. In otherembodiments, the retrovirus is replication-defective, e.g., having oneof more coding regions for the genes necessary for additional rounds ofvirion replication and packaging replaced with other genes, or deleted.

In one embodiment, the gRNA-encoding nucleic acid sequence is deliveredby a recombinant lentivirus. For example, the lentivirus isreplication-defective, e.g., does not comprise one or more genesrequired for viral replication.

In some embodiments, the gRNA-encoding nucleic acid sequence isdelivered by a recombinant adenovirus. In some embodiments, theadenovirus is engineered to have reduced immunity in human.

In some embodiments, the gRNA-encoding nucleic acid sequence isdelivered by a recombinant AAV. In some embodiments, the AAV does notincorporate its genome into that of a host cell, e.g., a target cell asdescribe herein. In some embodiments, the AAV can incorporate its genomeinto that of a host cell. In some embodiments, the AAV is aself-complementary adeno-associated virus (scAAV), e.g., a scAAV thatpackages both strands which anneal together to form double stranded DNA.

In one embodiment, an AAV capsid that can be used in the methodsdescribed herein is a capsid sequence from serotype AAV1, AAV2, AAV3,AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV.rh8, AAV.rh10, AAV.rh32/33,AAV.rh43, AAV.rh64R1, or AAV7m8.

In one embodiment, the gRNA-encoding DNA is delivered in a re-engineeredAAV capsid, e.g., with 50% or greater, e.g., 60% or greater, 70% orgreater, 80% or greater, 90% or greater, or 95% or greater, sequencehomology with a capsid sequence from serotypes AAV1, AAV2, AAV3, AAV4,AAV5, AAV6, AAV7, AAV8, AAV9, AAV.rh8, AAV.rh10, AAV.rh32/33, AAV.rh43,or AAV.rh64R1.

In one embodiment, the gRNA-encoding DNA is delivered by a chimeric AAVcapsid. Exemplary chimeric AAV capsids include, but are not limited to,AAV9i1, AAV2i8, AAV-DJ, AAV2G9, AAV2i8G9, or AAV8G9.

In one embodiment, the AAV is a self-complementary adeno-associatedvirus (scAAV), e.g., a scAAV that packages both strands which annealtogether to form double stranded DNA.

In some embodiments, the gRNA-encoding DNA is delivered by a hybridvirus, e.g., a hybrid of one or more of the viruses described herein. Inone embodiment, the hybrid virus is hybrid of an AAV (e.g., of any AAVserotype), with a Bocavirus, B19 virus, porcine AAV, goose AAV, felineAAV, canine AAV, or MVM.

A packaging cell is used to form a virus particle that is capable ofinfecting a target cell. Exemplary packaging cells include 293 cells,which can package adenovirus, and w2 or PA317 cells, which can packageretrovirus. A viral vector used in gene therapy is usually generated bya producer cell line that packages a nucleic acid vector into a viralparticle. The vector typically contains the minimal viral sequencesrequired for packaging and subsequent integration into a host or targetcell (if applicable). For example, an AAV vector used in gene therapytypically only possesses inverted terminal repeat (ITR) sequences fromthe AAV genome which are required for packaging and gene expression inthe host or target cell. The missing viral functions can be supplied intrans by the packaging cell line and/or plasmid containing E2A, E4, andVA genes from adenovirus, and plasmid encoding Rep and Cap genes fromAAV, as described in “Triple Transfection Protocol.” Henceforth, theviral DNA is packaged in a cell line, which contains a helper plasmidencoding the other AAV genes, namely rep and cap, but lacking ITRsequences. In certain embodiments, the viral DNA is packaged in aproducer cell line, which contains E1A and/or E1B genes from adenovirus.The cell line is also infected with adenovirus as a helper. The helpervirus (e.g., adenovirus or HSV) or helper plasmid promotes replicationof the AAV vector and expression of AAV genes from the helper plasmidwith ITRs. The helper plasmid is not packaged in significant amounts dueto a lack of ITR sequences. Contamination with adenovirus can be reducedby, e.g., heat treatment to which adenovirus is more sensitive than AAV.

In certain embodiments, the viral vector is capable of cell type and/ortissue type recognition. For example, the viral vector can bepseudotyped with a different/alternative viral envelope glycoprotein;engineered with a cell type-specific receptor (e.g., geneticmodification of the viral envelope glycoproteins to incorporatetargeting ligands such as a peptide ligand, single chain antibody, orgrowth factor); and/or engineered to have a molecular bridge with dualspecificities with one end recognizing a viral glycoprotein and theother end recognizing a moiety of the target cell surface (e.g.,ligand-receptor, monoclonal antibody, avidin-biotin and chemicalconjugation).

In certain embodiments, the viral vector achieves cell type specificexpression. For example, a tissue-specific promoter can be constructedto restrict expression of the transgene (gRNA) to only the target cell.The specificity of the vector can also be mediated by microRNA-dependentcontrol of transgene expression. In one embodiment, the viral vector hasincreased efficiency of fusion of the viral vector and a target cellmembrane. For example, a fusion protein such as fusion-competenthemagglutin (HA) can be incorporated to increase viral uptake intocells. In one embodiment, the viral vector has the ability of nuclearlocalization. For example, a virus that requires the breakdown of thenuclear envelope (during cell division) and therefore will not infect anon-diving cell can be altered to incorporate a nuclear localizationpeptide in the matrix protein of the virus thereby enabling thetransduction of non-proliferating cells.

In some embodiments, the gRNA-encoding DNA is delivered by a non-vectorbased method (e.g., using naked DNA or DNA complexes). For example, theDNA can be delivered, e.g., by organically modified silica or silicate(Ormosil), electroporation, transient cell compression or squeezing(see, e.g., Lee 2012), gene gun, sonoporation, magnetofection,lipid-mediated transfection, dendrimers, inorganic nanoparticles,calcium phosphates, or a combination thereof.

In one embodiment, delivery via electroporation comprises mixing thecells with the gRNA-encoding DNA in a cartridge, chamber or cuvette andapplying one or more electrical impulses of defined duration andamplitude. In one embodiment, delivery via electroporation is performedusing a system in which cells are mixed with the gRNA-encoding DNA in avessel connected to a device (e.g., a pump) which feeds the mixture intoa cartridge, chamber or cuvette wherein one or more electrical impulsesof defined duration and amplitude are applied, after which the cells aredelivered to a second vessel.

In some embodiments, the gRNA-encoding DNA is delivered by a combinationof a vector and a non-vector based method. For example, virosomescombine liposomes with an inactivated virus (e.g., HIV or influenzavirus), which can result in more efficient gene transfer, e.g., inrespiratory epithelial cells than either viral or liposomal methodsalone.

As described above, a nucleic acid may comprise a sequence encoding agRNA molecule comprising a targeting domain that is complementary with adesired target domain. In one embodiment, the nucleic acid molecule isan AAV vector. Exemplary AAV vectors that may be used in any of thedescribed compositions and methods include an AAV2 vector, a modifiedAAV2 vector, an AAV3 vector, a modified AAV3 vector, an AAV6 vector, amodified AAV6 vector, an AAV8 vector and an AAV9 vector. In yet anotherembodiment, the nucleic acid may further comprise a sequence thatencodes a second, third and/or fourth gRNA molecule as described herein.Each of the sequence encoding a gRNA molecule comprising a targetingdomain that is complementary with a desired target domain and thesequence that encodes a second, third and/or fourth gRNA molecule may bepresent on the same nucleic acid molecule, e.g., the same vector, e.g.,the same viral vector, e.g., the same adeno-associated virus (AAV)vector. In one embodiment, the nucleic acid molecule is an AAV vector.

In another embodiment, the sequence encoding a gRNA molecule comprisinga targeting domain that is complementary with a desired target domainand the sequence that encodes a second, third and/or fourth gRNAmolecule are on different vectors. For example, the sequence encoding agRNA molecule comprising a targeting domain that is complementary with adesired target domain may be present on a first nucleic acid molecule,e.g., a first vector, e.g., a first viral vector, e.g., a first AAVvector; and the sequence that encodes a second, third and/or fourth gRNAmolecule may be present on a second nucleic acid molecule, e.g., asecond vector, e.g., a second vector, e.g., a second AAV vector. In oneembodiment, the first and second nucleic acid molecules are AAV vectors.

In another embodiment, when a third and/or fourth gRNA molecule arepresent, each of the sequence encoding a gRNA molecule comprising atargeting domain that is complementary with a desired target domain andthe sequence that encodes a second, third and/or fourth gRNA moleculemay be present on the same nucleic acid molecule, e.g., the same vector,e.g., the same viral vector, e.g., an AAV vector. In one embodiment, thenucleic acid molecule is an AAV vector. In an alternate embodiment, eachof the sequence encoding a gRNA molecule comprising a targeting domainthat is complementary with a desired target domain and the sequence thatencodes a second, third and/or fourth gRNA molecule may be present onthe different nucleic acid molecules, e.g., different vectors, e.g., thedifferent viral vectors, e.g., different AAV vectors. In furtherembodiments, each of the sequence encoding a gRNA molecule comprising atargeting domain that is complementary with a desired target domain andthe sequence that encodes a second, third and/or fourth gRNA moleculemay be present on more than one nucleic acid molecule, but fewer thanfive nucleic acid molecules, e.g., AAV vectors.

The nucleic acids described herein may comprise a promoter operablylinked to the sequence that encodes the gRNA molecule of the sequenceencoding a gRNA molecule comprising a targeting domain that iscomplementary with a desired target domain, e.g., a promoter describedherein. The nucleic acid may further comprise a second promoter operablylinked to the sequence that encodes the second, third and/or fourth gRNAmolecule, e.g., a promoter described herein. The promoter and secondpromoter differ from one another. In one embodiment, the promoter andsecond promoter are the same.

In certain embodiments, the delivery vehicle is a non-viral vector, andin certain of these embodiments the non-viral vector is an inorganicnanoparticle. Exemplary inorganic nanoparticles include, e.g., magneticnanoparticles (e.g., Fe₃MnO₂) or silica. The outer surface of thenanoparticle can be conjugated with a positively charged polymer (e.g.,polyethylenimine, polylysine, polyserine) which allows for attachment(e.g., conjugation or entrapment) of payload. In one embodiment, thenon-viral vector is an organic nanoparticle (e.g., entrapment of thepayload inside the nanoparticle). Exemplary organic nanoparticlesinclude, e.g., SNALP liposomes that contain cationic lipids togetherwith neutral helper lipids which are coated with polyethylene glycol(PEG) and protamine and nucleic acid complex coated with lipid coating.

Exemplary lipids for gene transfer are shown below in Table 7.

TABLE 7 Lipids Used for Gene Transfer Lipid Abbreviation Feature1,2-Dioleoyl-sn-glycero-3-phosphatidylcholine DOPC Helper1,2-Dioleoyl-sn-glycero-3-phosphatidylethanolamine DOPE HelperCholesterol Helper N-[1-(2,3-Dioleyloxy)propyl]N,N,N-trimethylammoniumDOTMA Cationic chloride 1,2-Dioleoyloxy-3-trimethylammonium-propaneDOTAP Cationic Dioctadecylamidoglycylspermine DOGS CationicN-(3-Aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1- GAP-DLRIE Cationicpropanaminium bromide Cetyltrimethylammonium bromide CTAB Cationic6-Lauroxyhexyl ornithinate LHON Cationic1-(2,3-Dioleoyloxypropyl)-2,4,6-trimethylpyridinium 2Oc Cationic2,3-Dioleyloxy-N-[2(sperminecarboxamido-ethyl]- DOSPA CationicN,N-dimethyl-1-propanaminium trifluoroacetate1,2-Dioleyl-3-trimethylammonium-propane DOPA CationicN-(2-Hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1- MDRIE Cationicpropanaminium bromide Dimyristooxypropyl dimethyl hydroxy ethyl ammoniumbromide DMRI Cationic3β-[N-(N′,N′-Dimethylaminoethane)-carbamoyl]cholesterol DC-Chol CationicBis-guanidium-tren-cholesterol BGTC Cationic1,3-Diodeoxy-2-(6-carboxy-spermyl)-propylamide DOSPER CationicDimethyloctadecylammonium bromide DDAB CationicDioctadecylamidoglicylspermidin DSL Cationicrac-[(2,3-Dioctadecyloxypropyl)(2-hydroxyethyl)]- CLIP-1 Cationicdimethylammonium chloride rac-[2(2,3-Dihexadecyloxypropyl- CLIP-6Cationic oxymethyloxy)ethyl]trimethylammonium bromideEthyldimyristoylphosphatidylcholine EDMPC Cationic1,2-Distearyloxy-N,N-dimethyl-3-aminopropane DSDMA Cationic1,2-Dimyristoyl-trimethylammonium propane DMTAP CationicO,O′-Dimyristyl-N-lysyl aspartate DMKE Cationic1,2-Distearoyl-sn-glycero-3-ethylphosphocholine DSEPC CationicN-Palmitoyl D-erythro-sphingosyl carbamoyl-spermine CCS CationicN-t-Butyl-N0-tetradecyl-3-tetradecylaminopropionamidine diC14-amidineCationic Octadecenolyoxy[ethyl-2-heptadecenyl-3 hydroxyethyl] DOTIMCationic imidazolinium chlorideN1-Cholesteryloxycarbonyl-3,7-diazanonane-1,9-diamine CDAN Cationic2-(3-[Bis(3-amino-propyl)-amino]propylamino)-N- RPR209120 Cationicditetradecylcarbamoylme-ethyl-acetamide1,2-dilinoleyloxy-3-dimethylaminopropane DLinDMA Cationic2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane DLin-KC2- CationicDMA dilinoleyl-methyl-4-dimethylaminobutyrate DLin-MC3- Cationic DMA

Exemplary polymers for gene transfer are shown below in Table 8.

TABLE 8 Polymers Used for Gene Transfer Polymer AbbreviationPoly(ethylene)glycol PEG Polyethylenimine PEIDithiobis(succinimidylpropionate) DSPDimethyl-3,3′-dithiobispropionimidate DTBP Poly(ethylene imine)biscarbamate PEIC Poly(L-lysine) PLL Histidine modified PLLPoly(N-vinylpyrrolidone) PVP Poly(propylen imine) PPI Poly(amidoamine)PAMAM Poly(amido ethylenimine) SS-PAEI Triethylenetetramine TETAPoly(β-aminoester) Poly(4-hydroxy-L-proline ester) PHP Poly(allylamine)Poly(α-[4-aminobutyl]-L-glycolic acid) PAGA Poly(D,L-lactic-co-glycolicacid) PLGA Poly(A-ethyl-4-vinylpyridinium bromide) Poly(phosphazene)sPPZ Poly(phosphoester)s PPE Poly(phosphoramidate)s PPAPoly(N-2-hydroxypropylmethacrylamide) pHPMA Poly (2-(dimethylamino)ethylmethacrylate) pDMAEMA Poly(2-aminoethyl propylene phosphate) PPE-EAChitosan Galactosylated chitosan N-Dodacylated chitosan Histone CollagenDextran-spermine D-SPM

In one embodiment, the vehicle has targeting modifications to increasetarget cell update of nanoparticles and liposomes, e.g., cell specificantigens, monoclonal antibodies, single chain antibodies, aptamers,polymers, sugars (e.g., N-acetylgalactosamine (GalNAc)), and cellpenetrating peptides. In one embodiment, the vehicle uses fusogenic andendosome-destabilizing peptides/polymers. In one embodiment, the vehicleundergoes acid-triggered conformational changes (e.g., to accelerateendosomal escape of the cargo). In one embodiment, a stimuli-cleavablepolymer is used, e.g., for release in a cellular compartment. Forexample, disulfide-based cationic polymers that are cleaved in thereducing cellular environment can be used.

In one embodiment, the delivery vehicle is a biological non-viraldelivery vehicle. In one embodiment, the vehicle is an attenuatedbacterium (e.g., naturally or artificially engineered to be invasive butattenuated to prevent pathogenesis and expressing the transgene (e.g.,Listeria monocytogenes, certain Salmonella strains, Bifidobacteriumlongum, and modified Escherichia coli), bacteria having nutritional andtissue-specific tropism to target specific tissues, bacteria havingmodified surface proteins to alter target tissue specificity). In oneembodiment, the vehicle is a genetically modified bacteriophage (e.g.,engineered phages having large packaging capacity, less immunogenic,containing mammalian plasmid maintenance sequences and havingincorporated targeting ligands). In one embodiment, the vehicle is amammalian virus-like particle. For example, modified viral particles canbe generated (e.g., by purification of the “empty” particles followed byex vivo assembly of the virus with the desired cargo). The vehicle canalso be engineered to incorporate targeting ligands to alter targettissue specificity. In one embodiment, the vehicle is a biologicalliposome. For example, the biological liposome is a phospholipid-basedparticle derived from human cells (e.g., erythrocyte ghosts, which arered blood cells broken down into spherical structures derived from thesubject (e.g., tissue targeting can be achieved by attachment of varioustissue or cell-specific ligands), or secretory exosomes—subject (i.e.,patient) derived membrane-bound nanovesicle (30-100 nm) of endocyticorigin (e.g., can be produced from various cell types and can thereforebe taken up by cells without the need of for targeting ligands).

In one embodiment, one or more nucleic acid molecules (e.g., DNAmolecules) other than the components of a Cas system, e.g., the Cas9fusion molecule component and/or the gRNA molecule component describedherein, are delivered. In one embodiment, the nucleic acid molecule isdelivered at the same time as one or more of the gRNA molecule(s) aredelivered. In one embodiment, the nucleic acid molecule is deliveredbefore or after (e.g., less than about 30 minutes, 1 hour, 2 hours, 3hours, 6 hours, 9 hours, 12 hours, 1 day, 2 days, 3 days, 1 week, 2weeks, or 4 weeks) one or more of the gRNA molecule(s) are delivered. Inone embodiment, the nucleic acid molecule is delivered by a differentmeans than one or more of the gRNA molecule(s) are delivered. Thenucleic acid molecule can be delivered by any of the delivery methodsdescribed herein. For example, the nucleic acid molecule can bedelivered by a viral vector, e.g., an integration-deficient lentivirus,and the gRNA molecule component can be delivered by electroporation,e.g., such that the toxicity caused by nucleic acids (e.g., DNAs) can bereduced. In one embodiment, the nucleic acid molecule encodes atherapeutic protein, e.g., a protein described herein. In oneembodiment, the nucleic acid molecule encodes an RNA molecule, e.g., anRNA molecule described herein.

Delivery of RNA Encoding a gRNA Molecule

RNA encoding gRNA molecules can be delivered into cells, e.g., targetcells described herein, by art-known methods or as described herein. Forexample, gRNA-encoding RNA can be delivered, e.g., by microinjection,electroporation, transient cell compression or squeezing (see, e.g., Lee2012), lipid-mediated transfection, peptide-mediated delivery, or acombination thereof. gRNA-encoding RNA can be conjugated to moleculespromoting uptake by the target cells (e.g., target cells describedherein).

In one embodiment, delivery via electroporation comprises mixing thecells with the RNA encoding gRNA molecules in a cartridge, chamber orcuvette and applying one or more electrical impulses of defined durationand amplitude. In one embodiment, delivery via electroporation isperformed using a system in which cells are mixed with the RNA encodinggRNA molecules in a vessel connected to a device (e.g., a pump) whichfeeds the mixture into a cartridge, chamber or cuvette wherein one ormore electrical impulses of defined duration and amplitude are applied,after which the cells are delivered to a second vessel. gRNA-encodingRNA can be conjugated to molecules to promote uptake by the target cells(e.g., target cells described herein).

Delivery of Cas9 Polypeptides and Cas9 Fusion Molecules

Cas9 molecules and Cas9 fusion molecules can be delivered into cells byart-known methods or as described herein. For example, protein moleculescan be delivered, e.g., by microinjection, electroporation, transientcell compression or squeezing (see, e.g., Lee 2012), lipid-mediatedtransfection, peptide-mediated delivery, or a combination thereof.Delivery can be accompanied by DNA encoding a gRNA or by a gRNA. Cas9proteins and Cas9 fusion molecules can be conjugated to moleculespromoting uptake by the target cells (e.g., target cells describedherein).

In one embodiment, delivery via electroporation comprises mixing thecells with the Cas9 fusion molecules and/or gRNA molecules in acartridge, chamber or cuvette and applying one or more electricalimpulses of defined duration and amplitude. In one embodiment, deliveryvia electroporation is performed using a system in which cells are mixedwith the Cas9 fusion molecules and/or gRNA molecules in a vesselconnected to a device (e.g., a pump) which feeds the mixture into acartridge, chamber or cuvette wherein one or more electrical impulses ofdefined duration and amplitude are applied, after which the cells aredelivered to a second vessel. gRNA-encoding RNA can be conjugated tomolecules to promote uptake by the target cells (e.g., target cellsdescribed herein).

Route of Administration

Systemic modes of administration include oral and parenteral routes.Parenteral routes include, by way of example, intravenous, intramarrow,intrarterial, intramuscular, intradermal, subcutaneous, intranasal,inhalation, and intraperitoneal routes. Components administeredsystemically may be modified or formulated to target, e.g., HSCs,hematopoetic stem/progenitor cells, or erythroid progenitors orprecursor cells.

Local modes of administration include, by way of example, intramarrowinjection into the trabecular bone or intrafemoral injection into themarrow space, and infusion into the portal vein. In one embodiment,significantly smaller amounts of the components (compared with systemicapproaches) may exert an effect when administered locally (for example,directly into the bone marrow) compared to when administeredsystemically (for example, intravenously). Local modes of administrationcan reduce or eliminate the incidence of potentially toxic side effectsthat may occur when therapeutically effective amounts of a component areadministered systemically.

Administration may be provided as a periodic bolus (e.g., intravenously)or as continuous infusion from an internal reservoir or from an externalreservoir (for example, from an intravenous bag or implantable pump).Components may be administered locally, for example, by continuousrelease from a sustained release drug delivery device.

In addition, components may be formulated to permit release over aprolonged period of time. A release system can include a matrix of abiodegradable material or a material which releases the incorporatedcomponents by diffusion. The components can be homogeneously orheterogeneously distributed within the release system. A variety ofrelease systems may be useful, however, the choice of the appropriatesystem will depend upon rate of release required by a particularapplication. Both non-degradable and degradable release systems can beused. Suitable release systems include polymers and polymeric matrices,non-polymeric matrices, or inorganic and organic excipients and diluentssuch as, but not limited to, calcium carbonate and sugar (for example,trehalose). Release systems may be natural or synthetic. However,synthetic release systems are preferred because generally they are morereliable, more reproducible and produce more defined release profiles.The release system material can be selected so that components havingdifferent molecular weights are released by diffusion through ordegradation of the material.

Representative synthetic, biodegradable polymers include, for example:polyamides such as poly(amino acids) and poly(peptides); polyesters suchas poly(lactic acid), poly(glycolic acid), poly(lactic-co-glycolicacid), and poly(caprolactone); poly(anhydrides); polyorthoesters;polycarbonates; and chemical derivatives thereof (substitutions,additions of chemical groups, for example, alkyl, alkylene,hydroxylations, oxidations, and other modifications routinely made bythose skilled in the art), copolymers and mixtures thereof.Representative synthetic, non-degradable polymers include, for example:polyethers such as poly(ethylene oxide), poly(ethylene glycol), andpoly(tetramethylene oxide); vinyl polymers-polyacrylates andpolymethacrylates such as methyl, ethyl, other alkyl, hydroxyethylmethacrylate, acrylic and methacrylic acids, and others such aspoly(vinyl alcohol), poly(vinyl pyrolidone), and poly(vinyl acetate);poly(urethanes); cellulose and its derivatives such as alkyl,hydroxyalkyl, ethers, esters, nitrocellulose, and various celluloseacetates; polysiloxanes; and any chemical derivatives thereof(substitutions, additions of chemical groups, for example, alkyl,alkylene, hydroxylations, oxidations, and other modifications routinelymade by those skilled in the art), copolymers and mixtures thereof.

Poly(lactide-co-glycolide) microsphere can also be used for injection.Typically the microspheres are composed of a polymer of lactic acid andglycolic acid, which are structured to form hollow spheres. The spherescan be approximately 15-30 microns in diameter and can be loaded withcomponents described herein.

Bi-Modal or Differential Delivery of Components

Separate delivery of the components of a Cas system, e.g., the Cas9fusion molecule component and the gRNA molecule component, and moreparticularly, delivery of the components by differing modes, can enhanceperformance, e.g., by improving tissue specificity and safety.

In one embodiment, the Cas9 fusion molecule and the gRNA molecule aredelivered by different modes, or as sometimes referred to herein asdifferential modes. Different or differential modes, as used herein,refer modes of delivery that confer different pharmacodynamic orpharmacokinetic properties on the subject component molecule, e.g., aCas9 fusion molecule, gRNA molecule, or payload. For example, the modesof delivery can result in different tissue distribution, differenthalf-life, or different temporal distribution, e.g., in a selectedcompartment, tissue, or organ.

Some modes of delivery, e.g., delivery by a nucleic acid vector thatpersists in a cell, or in progeny of a cell, e.g., by autonomousreplication or insertion into cellular nucleic acid, result in morepersistent expression of and presence of a component. Examples includeviral, e.g., AAV or lentivirus, delivery.

By way of example, the components, e.g., a Cas9 fusion molecule and agRNA molecule, can be delivered by modes that differ in terms ofresulting half-life or persistent of the delivered component the body,or in a particular compartment, tissue or organ. In one embodiment, agRNA molecule can be delivered by such modes. The Cas9 fusion moleculecomponent can be delivered by a mode which results in less persistenceor less exposure to the body or a particular compartment or tissue ororgan.

More generally, in one embodiment, a first mode of delivery is used todeliver a first component and a second mode of delivery is used todeliver a second component. The first mode of delivery confers a firstpharmacodynamic or pharmacokinetic property. The first pharmacodynamicproperty can be, e.g., distribution, persistence, or exposure, of thecomponent, or of a nucleic acid that encodes the component, in the body,a compartment, tissue or organ. The second mode of delivery confers asecond pharmacodynamic or pharmacokinetic property. The secondpharmacodynamic property can be, e.g., distribution, persistence, orexposure, of the component, or of a nucleic acid that encodes thecomponent, in the body, a compartment, tissue or organ.

In certain embodiments, the first pharmacodynamic or pharmacokineticproperty, e.g., distribution, persistence or exposure, is more limitedthan the second pharmacodynamic or pharmacokinetic property.

In certain embodiments, the first mode of delivery is selected tooptimize, e.g., minimize, a pharmacodynamic or pharmacokinetic property,e.g., distribution, persistence or exposure.

In certain embodiments, the second mode of delivery is selected tooptimize, e.g., maximize, a pharmacodynamic or pharmacokinetic property,e.g., distribution, persistence or exposure.

In one embodiments, the first mode of delivery comprises the use of arelatively persistent element, e.g., a nucleic acid, e.g., a plasmid orviral vector, e.g., an AAV or lentivirus. As such vectors are relativelypersistent product transcribed from them would be relatively persistent.

In certain embodiments, the second mode of delivery comprises arelatively transient element, e.g., an RNA or protein.

In certain embodiments, the first component comprises gRNA molecule, andthe delivery mode is relatively persistent, e.g., the gRNA istranscribed from a plasmid or viral vector, e.g., an AAV or lentivirus.Transcription of these genes would be of little physiologicalconsequence because the genes do not encode for a protein product, andthe gRNAs are incapable of acting in isolation. The second component, aCas9 fusion molecule, is delivered in a transient manner, for example asprotein, ensuring that the full Cas9 molecule/gRNA molecule complex isonly present and active for a short period of time.

Furthermore, the components can be delivered in different molecular formor with different delivery vectors that complement one another toenhance safety and tissue specificity.

Use of differential delivery modes can enhance performance, safetyand/or efficacy, e.g., the likelihood of an eventual off-targetmodification can be reduced. Delivery of immunogenic components, e.g.,Cas9 fusion molecules, by less persistent modes can reduceimmunogenicity, as peptides from the bacterially-derived Cas enzyme aredisplayed on the surface of the cell by MHC molecules. A two-partdelivery system can alleviate these drawbacks.

Differential delivery modes can be used to deliver components todifferent, but overlapping target regions. The formation active complexis minimized outside the overlap of the target regions. Thus, in oneembodiment, a first component, e.g., a gRNA molecule is delivered by afirst delivery mode that results in a first spatial, e.g., tissue,distribution. A second component, e.g., a Cas9 fusion molecule isdelivered by a second delivery mode that results in a second spatial,e.g., tissue, distribution. In one embodiment, the first mode comprisesa first element selected from a liposome, nanoparticle, e.g., polymericnanoparticle, and a nucleic acid, e.g., viral vector. The second modecomprises a second element selected from the group. In one embodiment,the first mode of delivery comprises a first targeting element, e.g., acell specific receptor or an antibody, and the second mode of deliverydoes not include that element. In certain embodiments, the second modeof delivery comprises a second targeting element, e.g., a second cellspecific receptor or second antibody.

When the Cas9 fusion molecule is delivered in a liposome, or polymericnanoparticle, there is the potential for delivery to and therapeuticactivity in multiple tissues, when it may be desirable to only target asingle tissue. A two-part delivery system can resolve this challenge andenhance tissue specificity. If the gRNA molecule and the Cas9 fusionmolecule are packaged in separated delivery vehicles with distinct butoverlapping tissue tropism, the fully functional complex is only beformed in the tissue that is targeted by both vectors.

Disclosed herein are methods of altering a cell, e.g., altering thestructure, e.g., altering the sequence, of a target nucleic acid of acell, comprising contacting said cell with: (a) a gRNA molecule thattargets the target gene, e.g., a gRNA molecule as described herein; (b)a Cas9 fusion molecule, e.g., a Cas9 fusion molecule as describedherein; and optionally, (c) a second, third and/or fourth gRNA thattargets the target gene, e.g., a gRNA molecule; as described herein. Inone embodiment, the method comprises contacting said cell with (a) and(b). In one embodiment, the method comprises contacting said cell with(a), (b), and (c). The targeting domain of the gRNA molecule of (a) andoptionally (c) may be selected from a targeting domain sequencedescribed herein.

In one embodiment, the method comprises contacting a cell from a subjectsuffering from or likely to develop a disease. The cell may be from asubject having a mutation at a target position in a target gene. In oneembodiment, the cell being contacted in the disclosed method is anerythroid cell. The contacting may be performed ex vivo and thecontacted cell may be returned to the subject's body after thecontacting step. In another embodiment, the contacting step may beperformed in vivo. In one embodiment, the method of altering a cell asdescribed herein comprises acquiring knowledge of the sequence at atarget position in said cell, prior to the contacting step. Acquiringknowledge of the sequence at a target position in the cell may be bysequencing the target gene, or a portion of the target gene. In oneembodiment, the contacting step of the method comprises contacting thecell with a nucleic acid, e.g., a vector, e.g., an AAV vector, thatexpresses at least one of (a), (b), and (c). In one embodiment, thecontacting step of the method comprises contacting the cell with anucleic acid, e.g., a vector, e.g., an AAV vector, that expresses eachof (a), (b), and (c). In another embodiment, the contacting step of themethod comprises delivering to the cell a Cas9 fusion molecule of (b)and a nucleic acid which encodes a gRNA molecule (a) and optionally, asecond gRNA molecule (c)(i) (and further optionally, a third gRNAmolecule (c)(iv) and/or fourth gRNA molecule (c)(iii).

In one embodiment, contacting comprises contacting the cell with anucleic acid, e.g., a vector, e.g., an AAV vector, e.g., an AAV2 vector,a modified AAV2 vector, an AAV3 vector, a modified AAV3 vector, an AAV6vector, a modified AAV6 vector, an AAV8 vector or an AAV9 vector.

In one embodiment, contacting comprises delivering to the cell a Cas9fusion molecule of (b), as a protein, and a nucleic acid which encodes(a) and optionally a second, third and/or fourth gRNA molecule of (c).

In one embodiment, contacting comprises delivering to the cell a Cas9fusion molecule of (b), as a protein, said gRNA molecule of (a), as anRNA, and optionally said second, third and/or fourth gRNA molecule of(c), as an RNA.

When the method comprises correcting the mutation at a target positionby HDR, a Cas9 fusion molecule of (b), at least one gRNA molecule, e.g.,a gRNA molecule of (a) are included in the contacting step. In oneembodiment, a cell of the subject is contacted ex vivo with (a), (b),and optionally (c)(i), further optionally (c)(ii), and still furtheroptionally (c)(iii). In another embodiment, said cell is returned to thesubject's body. In one embodiment, a cell of the subject is contacted isin vivo with (a), (b), and optionally (c)(i), further optionally(c)(ii), and still further optionally (c)(iii). In one embodiment, thecell of the subject is contacted in vivo by intravenous delivery of (a),(b), and optionally (c)(i), further optionally (c)(ii), and stillfurther optionally (c)(iii). In one embodiment, the cell of the subjectis contacted in vivo by intramuscular delivery of (a), (b), andoptionally (c)(i), further optionally (c)(ii), and still furtheroptionally (c)(iii). In one embodiment, the cell of the subject iscontacted in vivo by subcutaneous delivery of (a), (b), and optionally(c)(i), further optionally (c)(ii), and still further optionally(c)(iii). In one embodiment, the cell of the subject is contacted invivo by intra-bone marrow (IBM) delivery of (a), (b), and optionally(c)(i), further optionally (c)(ii), and still further optionally(c)(iii).

In one embodiment, contacting comprises contacting the subject with anucleic acid, e.g., a vector, e.g., an AAV vector, described herein,e.g., a nucleic acid that encodes at least one of (a), (b), andoptionally (c)(i), further optionally (c)(ii), and still furtheroptionally (c)(iii).

In one embodiment, contacting comprises delivering to said subject saidCas9 fusion molecule of (b), as a protein, and a nucleic acid whichencodes (a), and optionally (c)(i), further optionally (c)(ii), andstill further optionally (c)(iii).

In one embodiment, contacting comprises delivering to the subject theCas9 fusion molecule of (b), as a protein, the gRNA molecule of (a), asan RNA, and optionally the second, third and/or fourth gRNA molecule of(c), as an RNA.

In one embodiment, a cell of the subject is contacted ex vivo with (a),(b) and optionally (c)(i), further optionally (c)(ii), and still furtheroptionally (c)(iii). In one embodiment, said cell is returned to thesubject's body.

In one embodiment, a cell of the subject is contacted is in vivo with(a), (b) and optionally (c)(i), further optionally (c)(ii), and stillfurther optionally (c)(iii). In one embodiment, the cell of the subjectis contacted in vivo by intravenous delivery of (a), (b) and optionally(c)(i), further optionally (c)(ii), and still further optionally(c)(iii). In one embodiment, the cell of the subject is contacted invivo by intramuscular delivery of (a), (b) and optionally (c)(i),further optionally (c)(ii), and still further optionally (c)(iii). Inone embodiment, the cell of the subject is contacted in vivo bysubcutaneous delivery of (a), (b) and optionally (c)(i), furtheroptionally (c)(ii), and still further optionally (c)(iii). In oneembodiment, the cell of the subject is contacted in vivo by intra-bonemarrow (IBM) delivery of (a), (b) and optionally (c)(i), furtheroptionally (c)(ii), and still further optionally (c)(iii).

In one embodiment, contacting comprises contacting the subject with anucleic acid, e.g., a vector, e.g., an AAV vector, described herein,e.g., a nucleic acid that encodes at least one of (a), (b), andoptionally (c)(i), further optionally (c)(ii), and still furtheroptionally (c)(iii).

In one embodiment, contacting comprises delivering to said subject saidCas9 fusion molecule of (b), as a protein, and a nucleic acid whichencodes (a) and optionally (c)(i), further optionally (c)(ii), and stillfurther optionally (c)(iii).

In one embodiment, contacting comprises delivering to the subject theCas9 fusion molecule of (b), as a protein, the gRNA molecule of (a), asan RNA, and optionally the second, third and/or fourth gRNA molecule of(c), as an RNA.

In one embodiment, disclosed herein are kits comprising compositions ofthe invention and instructions for use.

Ex Vivo Delivery

In some embodiments, components described in Table 5 are introduced intocells which are then introduced into the subject. Methods of introducingthe components can include, e.g., any of the delivery methods describedin Table 6.

Modified Nucleosides, Nucleotides, and Nucleic Acids

Modified nucleosides and modified nucleotides can be present in nucleicacids, e.g., particularly gRNA molecule, but also other forms of RNA,e.g., mRNA, RNAi, or siRNA. As described herein, “nucleoside” is definedas a compound containing a five-carbon sugar molecule (a pentose orribose) or derivative thereof, and an organic base, purine orpyrimidine, or a derivative thereof. As described herein, “nucleotide”is defined as a nucleoside further comprising a phosphate group.

Modified nucleosides and nucleotides can include one or more of:

(i) alteration, e.g., replacement, of one or both of the non-linkingphosphate oxygens and/or of one or more of the linking phosphate oxygensin the phosphodiester backbone linkage;

(ii) alteration, e.g., replacement, of a constituent of the ribosesugar, e.g., of the 2′ hydroxyl on the ribose sugar;

(iii) wholesale replacement of the phosphate moiety with “dephospho”linkers;

(iv) modification or replacement of a naturally occurring nucleobase;

(v) replacement or modification of the ribose-phosphate backbone;

(vi) modification of the 3′ end or 5′ end of the oligonucleotide, e.g.,removal, modification or replacement of a terminal phosphate group orconjugation of a moiety; and

(vii) modification of the sugar.

The modifications listed above can be combined to provide modifiednucleosides and nucleotides that can have two, three, four, or moremodifications. For example, a modified nucleoside or nucleotide can havea modified sugar and a modified nucleobase. In one embodiment, everybase of a gRNA is modified, e.g., all bases have a modified phosphategroup, e.g., all are phosphorothioate groups. In one embodiment, all, orsubstantially all, of the phosphate groups of a unimolecular (orchimeric) or modular gRNA molecule are replaced with phosphorothioategroups.

In one embodiment, modified nucleotides, e.g., nucleotides havingmodifications as described herein, can be incorporated into a nucleicacid, e.g., a “modified nucleic acid.” In one embodiment, the modifiednucleic acids comprise one, two, three or more modified nucleotides. Inone embodiment, at least 5% (e.g., at least about 5%, at least about10%, at least about 15%, at least about 20%, at least about 25%, atleast about 30%, at least about 35%, at least about 40%, at least about45%, at least about 50%, at least about 55%, at least about 60%, atleast about 65%, at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 95%, orabout 100%) of the positions in a modified nucleic acid are a modifiednucleotides.

Unmodified nucleic acids can be prone to degradation by, e.g., cellularnucleases. For example, nucleases can hydrolyze nucleic acidphosphodiester bonds. Accordingly, in one aspect the modified nucleicacids described herein can contain one or more modified nucleosides ornucleotides, e.g., to introduce stability toward nucleases.

In one embodiment, the modified nucleosides, modified nucleotides, andmodified nucleic acids described herein can exhibit a reduced innateimmune response when introduced into a population of cells, both in vivoand ex vivo. The term “innate immune response” includes a cellularresponse to exogenous nucleic acids, including single stranded nucleicacids, generally of viral or bacterial origin, which involves theinduction of cytokine expression and release, particularly theinterferons, and cell death. In one embodiment, the modifiednucleosides, modified nucleotides, and modified nucleic acids describedherein can disrupt binding of a major groove interacting partner withthe nucleic acid. In one embodiment, the modified nucleosides, modifiednucleotides, and modified nucleic acids described herein can exhibit areduced innate immune response when introduced into a population ofcells, both in vivo and ex vivo, and also disrupt binding of a majorgroove interacting partner with the nucleic acid.

miRNA Binding Sites

microRNAs (or miRNAs) are naturally occurring cellular 19-25 nucleotidelong noncoding RNAs. They bind to nucleic acid molecules having anappropriate miRNA binding site, e.g., in the 3′ UTR of an mRNA, anddown-regulate gene expression. While not wishing to be bound by theoryit is believed that this down regulation occurs either by reducingnucleic acid molecule stability or by inhibiting translation. An RNAspecies disclosed herein, e.g., an mRNA encoding Cas9 can comprise anmiRNA binding site, e.g., in its 3′UTR. The miRNA binding site can beselected to promote down regulation of expression is a selected celltype. By way of example, the incorporation of a binding site formiR-122, a microRNA abundant in liver, can inhibit the expression of thegene of interest in the liver.

EXAMPLES

The following Examples are merely illustrative and are not intended tolimit the scope or content of the invention in any way.

Example 1: Constructing Single- and Multi-Cysteine Variant Cas9 Proteins

To generate single- and multi-cysteine variant Cas9 proteins,established molecular biology techniques and recombinant DNA proceduresknown to the ordinarily skilled artisan are used. A nucleotide sequenceencoding the protein sequence of Streptococcus pyogenes Cas9 (SpCas9)and Staphylococcus aureus Cas9 (SaCas9) are modified using site-directedmutagenesis based, in part, on the published crystal structures ofSpCas9 and SaCas9, as described in, e.g., in Anders et al., 2014 Nature513(7519): 569-73; Nishimau et al. Cell, 162(5): 1113-26, to producesingle- and multi-cysteine variant Cas9 proteins. For example, usingsite-directed mutagenesis, the protein sequence of SpCas9 is firstmutated to replace native cysteine residues at positions 80 and 574 withserine (i.e., C80S and C574S). Similarly, using site-directedmutagenesis, the protein sequence of SaCas9 is first mutated to replacenative cysteine residues at positions 237, 534 and 946 with serine(i.e., C237S, C534 and C946S). It is known that these conservative pointmutations do not abolish Cas9 activity; see, e.g., Nishimau et al. Cell,162(5): 1113-26. Site-directed mutagenesis is then performed to generatesingle- and multi-cysteine variant Cas9 proteins. Exemplary cysteinevariant Cas9 proteins are provided below:

Plasmid Mutation(s) Species Description pJZ001 C80S C574S PyogenesSpCas9 no cys variant pJZ002 C80S Pyogenes SpCas9 cys variant pJZ003C80S C574S D147C Pyogenes SpCas9 cys variant pJZ004 C80S_C574S S204CPyogenes SpCas9 cys variant pJZ005 C80S C574S Q228C Pyogenes SpCas9 cysvariant pJZ006 C80S_C574S N235C Pyogenes SpCas9 cys variant pJZ007C80S_C574S D257C Pyogenes SpCas9 cys variant pJZ008 C80S_C574S D284CPyogenes SpCas9 cys variant pJZ009 C80S C574S T313C Pyogenes SpCas9 cysvariant pJZ010 C80S C574S D326C Pyogenes SpCas9 cys variant pJZ011 C80SC574S D384C Pyogenes SpCas9 cys variant pJZ012 C80S C574S D428C PyogenesSpCas9 cys variant pJZ013 C80S_C574S N504C Pyogenes SpCas9 cys variantpJZ014 C80S C574S R535C Pyogenes SpCas9 cys variant pJZ015 C80S C574SL551C Pyogenes SpCas9 cys variant pJZ016 C80S C574S N556C PyogenesSpCas9 cys variant pJZ017 C80S C574S K558C Pyogenes SpCas9 cys variantpJZ018 C80S C574S E566C Pyogenes SpCas9 cys variant pJZ019 C80S_C574SD567C Pyogenes SpCas9 cys variant pJZ020 C80S C574S T605C PyogenesSpCas9 cys variant pJZ021 C80S C574S T638C Pyogenes SpCas9 cys variantpJZ022 C80S C574S A640C Pyogenes SpCas9 cys variant pJZ023 C80S C574SQ674C Pyogenes SpCas9 cys variant pJZ024 C80S C574S E945C PyogenesSpCas9 cys variant pJZ025 C80S C574S N946C Pyogenes SpCas9 cys variantpJZ026 C80S C574S L1004C Pyogenes SpCas9 cys variant pJZ027 C80S C574ST1065C Pyogenes SpCas9 cys variant pJZ028 C80S C574S K1076C PyogenesSpCas9 cys variant pJZ029 C80S C574S D1117C Pyogenes SpCas9 cys variantpJZ030 C80S C574S S1154C Pyogenes SpCas9 cys variant pJZ031 C80S C574SD1328C Pyogenes SpCas9 cys variant pJZ037 C237S C534S C946A AureusSaCas9 no cys variant pJZ038 D147C D384C N556C Pyogenes SpCas9 multi-cysA640C E945C variant pJZ039 Q192C S431C E745C Aureus SaCas9 multi-cysD795C D849C variant pJZ040 C237S C534S C946A Aureus SaCas9 cys variantQ192C pJZ041 C237S C534S C946A Aureus SaCas9 cys variant S431C pJZ042C237S C534S C946A Aureus SaCas9 cys variant E745C pJZ043 C237S C534SC946A Aureus SaCas9 cys variant D795C pJZ044 C237S C534S C946A AureusSaCas9 cys variant D849C

For expression in bacteria, cultured cells, or animal tissues, thenucleotide sequence encoding the single- and multi-cysteine variant Cas9proteins is operably linked to one or more transcriptional controlelements, e.g., promoter and/or enhancer elements, which enableexpression in the relevant bacteria, cultured cells, or animal tissue.The single- and multi-cysteine variant Cas9 proteins can be purifiedfrom the bacteria, cultured cells, or animal tissue using establishedbiochemical techniques. To generate mRNA encoding the single- andmulti-cysteine variant Cas9 proteins, the nucleotide sequence encodingthe single- and multi-cysteine variant Cas9 proteins is operably linkedto a promoter, e.g., a bacteriophage promoter, e.g., a T7 RNA polymerasepromoter enabling in vitro transcription of mRNA encoding the single-and multi-cysteine variant Cas9 proteins.

The single- and multi-cysteine variant Cas9 proteins allow forsite-specific bioconjugation to other molecular species, as describedfurther herein.

Example 2: Generation of Cas9 Fusion Molecules by Covalent Attachment ofSingle- and Multi-Cysteine Variant Cas9 Proteins to Exogenous DonorTemplate Sequence Using 5′-Maleimide-Modified Exogenous Donor TemplateSequence

To generate Cas9 fusion molecules, the single- and multi-cysteinevariant Cas9 proteins described in Example 1 are covalently attached totemplate nucleic acid using a 5′-maleimide-modified exogenous donortemplate sequence (FIG. 1). Generally, the template nucleic acid isprepared such that the 5′-end of the template nucleic acid is modifiedto include maleimide modification at its 5′-end. The maleimidemodification may have a one, or two or more carbon spacer arm betweenthe 5′-end of the template nucleic acid and the maleimide modificationfor purposes of reducing steric interactions between the maleimidemodification and the template nucleic acid. The 5′-maleimide-modifiedtemplate nucleic acid is then incubated in the presence of a single-and/or multi-cysteine variant Cas9 protein molecule described in Example1, at 4° C. overnight or at room temperature for 4 hours in buffercontaining TCEP (tris(2-carboxyethyl)phosphine) to keep free cysteinesreduced and prevent disulfide bonds from forming, according to standardprocedures known to the ordinarily skilled artisan, to covalently attachthe 5′-maleimide-modified template nucleic acid to at least one thiolgroup (e.g., a surface exposed thiol group) from the single- and/ormulti-cysteine variant Cas9 protein molecule described in Example 1, togenerate a Cas9 fusion molecule (FIG. 1).

To test the efficiency of the Cas9 fusion molecule prepared as describedin this Example, the reaction products are analyzed using anelectrophoretic mobility shift assay using standard procedures known tothe ordinary skilled artisan. Results provided in FIG. 2 indicate thegeneration of Cas9 fusion molecule as described in this Example.

Example 3: Generation of Cas9 Fusion Molecules by Covalent Attachment ofa Cas9 Protein to Template Nucleic Acid Via HyNic-4FB Conjugation

To generate Cas9 fusion molecules, a HyNic-modified Cas9 protein iscovalently attached to a 5′-4-Formylbenzamide (4FB)-modified templatenucleic acid. The template nucleic acid is prepared such that the 5′-endof the template nucleic acid is modified to include a5′-4-formylbenzamide (4FB) moiety at its 5′-end (TriLinkBioTechnologies). The chemistry that couples the Cas9 protein to the4FB-modified template nucleic acid takes advantage of free primary aminegroups that are present naturally (or are present as a consequence ofsite-directed mutagenesis) on the protein surface. Specifically, a Cas9protein is incubated in the presence ofsuccinimidyl-6-hydrazino-nicotinamide (S-HyNic) (Solulink), which reactswith at least one primary amine on the Cas9 protein to form a6-hydrazino-nicotinamide (HyNic)-modified Cas9 molecule. The6-hydrazino-nicotinamide (HyNic)-modified Cas9 protein molecule is thenincubated with the 5′-(4FB)-modified template nucleic acid to generateCas9 fusion molecules. Assays to test the efficiency of the Cas9 fusionmolecule prepared as described in this Example can be performed byanalyzing the reaction products via an electrophoretic mobility shiftassay using standard procedures known to the ordinary skilled artisan.

Example 4: Generation of Cas9 Fusion Molecules by Reaction of aCas9-HaloTag Protein Molecule with Haloalkane-Modified Template NucleicAcid

To generate Cas9 fusion molecules, a Cas9-HaloTag protein molecule iscreated by expressing a nucleic acid construct encoding a HaloTag,optionally fused to a nucleic acid sequence encoding a linker sequence,fused either to the N-terminus or C-terminus of a nucleic acid sequenceencoding a Cas9 protein molecule. The nucleic acid construct encoding aHaloTag, is a variant nucleic acid sequence that encodes for a mutantHaloTag comprising a H272F mutation. The HaloTag variant protein (i.e.,the H272F HaloTag protein) facilitates the formation of a covalent bondbetween the HaloTag variant and a haloalkane-modified nucleic acid,e.g., a bromoalkane-modified template nucleic acid.

Generally, for expression in bacteria, cultured cells, or animaltissues, the nucleotide sequence encoding the Cas9 protein fusion (i.e.,a Cas9 protein molecule fused to a HaloTag variant) is operably linkedto one or more transcriptional control elements, e.g., promoter and/orenhancer elements, which enable expression in the relevant bacteria,cultured cells, or animal tissue. The Cas9 protein fusion can bepurified from the bacteria, cultured cells, or animal tissue usingestablished biochemical techniques. Exemplary Cas9 protein fusions(i.e., a Cas9 protein molecule fused to a HaloTag variant) are providedbelow:

TABLR 9  Cas9 protein Linker sequence fusion (AA) AbbreviationHaloTag-XTEN  SGSETPGTSESATPES HXC linker-Cas9 HaloTag-GGS9GGSGGSGGSGGSGGSG HGC linker-Cas9 GSGGSGGSGGS Cas9-XTEN SGSETPGTSESATPESCXH linker-HaloTag Cas9-GGS9  GGSGGSGGSGGSGGSG CGH linker-HaloTagGSGGSGGSGGS

The effect of covalent attachment of the template sequence to Cas9 onthe frequency of gene correction (HDR) was examined. Generally, the Cas9protein fusion molecules of Table 9 were prepared as follows.

Cloning: The Cas9 gene and the HaloTag gene were generated by genesynthesis and cloned into a bacterial expression vector (using standardmolecular cloning techniques) with one of the linker sequences providedin Table 9 (i.e., the XTEN or the GGS₉ linker) in the arrangements setforth in Table 9. This resulted in 4 unique nucleic acid sequences forexpressing the Cas9 protein fusion molecules of Table 9. All fourconstructs comprise an N-terminal His-tag, with sequence His₆, and anuclear localization signal (NLS), with sequence PKKKRKV. In alternativeembodiments, the His₆-tag and the NLS can be cloned to the C-terminus ofthe Cas9 protein fusion molecules. In alternative embodiments, the Cas9gene sequence from either S. Pyogenes (spcas9) or from S. aureus(sacas9) may also be used.

Pre-conjugation of template sequence to a Cas9-HaloTag protein fusion:Cas9-HaloTag protein fusions (i.e., HXC, HGC, CXH, and CGH, see Table 9)were each incubated with a bromohexyl (BrH)-labeled 30 nucleotide (30mer) nucleic acid sequence for 1 hour at 37° C., to form a Cas9-HaloTagprotein fusion that is conjugated to the 30 nucleotide nucleic acidsequence (FIGS. 4A and 4B). A 149 nucleotide nucleic acid sequence (149mer) was subsequently annealed to the nucleic acid portion of theCas9-HaloTag protein fusion that is conjugated to the 30 nucleotidenucleic acid sequence by incubating the 149 mer with the Cas9-HaloTagprotein fusion that is conjugated to the 30 nucleotide nucleic acidsequence in the presence of a DNA splint, followed by addition of a T4DNA ligase, incubating the reaction at room temperature for 1 hour (FIG.4). The reaction resulted in a Cas9-HaloTag fusion molecule covalentlyattached to a 179 nucleotide template sequence (FIG. 4). A CC8 guidemolecule was subsequently added to the reaction mixture, which was thenallowed to incubate for 15 minutes prior to nucleofecting into U2OScells, according to standard procedures known to one of skill in theart. Four days after nucleofection, genomic DNA was extracted and atarget sequence locus was amplified and analyzed by next-generationsequencing and scored for % HDR (see FIG. 5).

Conjugation of pre-annealed template sequence to a Cas9-HaloTag proteinfusion: The 149 mer was pre-annealed to a DNA splint. The BrH-labeled 30mer was subsequently ligated to the pre-annealed 149 mer—DNA splintcomplex using T4 DNA ligase. The ligation reaction was performed at roomtemperature for 1 hour, resulting in a full length template sequence of179 nucleotides (i.e., 179 mer). The 179 mer was added to a reactionwith a Cas9-HaloTag protein fusion (i.e., HXC, HGC, CXH, or CGH) andincubated for 1 hour at 37° C., resulting in a Cas9-HaloTag fusionmolecule covalently attached to a 179 nucleotide template sequence (FIG.4). The CC8 guide was then added to this mixture and incubated for 15minutes prior to nucleofecting into U2OS cells, according to standardprocedures known to one of skill in the art. Four days afternucleofection, genomic DNA was extracted and a target sequence locus wasamplified and analyzed by next-generation sequencing and scored for %HDR (FIG. 5).

Results: HDR efficiency can be assayed using a standard nuclofectionassay in U2OS cells by comparing the results using the Cas9 fusionmolecules (i.e., Cas9-HaloTag protein fusion—template nucleic acidconjugates) in comparison the Cas9-HaloTag protein fusion without aconjugated template nucleic acid. The results in FIG. 5 demonstrate thatthe Cas9 fusion molecules (i.e., Cas9-HaloTag protein fusion—templatenucleic acid conjugates) increased the HDR efficiency compared toreactions performed with unconjugated template nucleic acid.Specifically, the results demonstrated a higher rate of HDR as detectedby sequencing when the cells were nucleofected with the Cas9 fusionmolecule HGC using the pre-conjugation method for conjugating thetemplate nucleic acid to the Cas9-HaloTag protein fusion. Thus, therecruitment of the template nucleic acid to the Cas9 via covalentattachment resulted in a 4% increase in HDR gene correction (FIG. 5).The results also demonstrated that the conjugation method using apre-annealed full length 179 mer does not yield significant conjugationproducts to the Halo-Cas9 protein fusions tested. Successful conjugationwas only observed via the “pre-conjugation” method, by first conjugatingthe BrH-labeled 30 mer to the Halo-Cas9 protein fusion (FIG. 4A)followed by subsequent splint ligation using T4 DNA ligase and the 149mer.

Example 5: Generation of Cas9 Fusion Molecules by Covalent Attachment ofSingle- and Multi-Cysteine Variant Cas9 Proteins to Template NucleicAcid Using Acrydite-Modified Template Nucleic Acid

To generate Cas9 fusion molecules, the single- and multi-cysteinevariant Cas9 proteins described in Example 1 are covalently attached totemplate nucleic acid using an acrydite-modified template nucleic acid.The template nucleic acid is prepared such that the 5′-end of thetemplate nucleic acid is modified to include an acrydite moiety at its5′-end. The template nucleic acid may also be synthesized such that the3′-end of the template nucleic acid is modified to include an acryditemoiety either at its 3′-end. The template nucleic acid may also besynthesized such that an internal nucleic acid residue of the templatenucleic acid is modified to include an acrydite moiety either at aninternal nucleic acid residue. The acrydite-modified template nucleicacid is then incubated in the presence of a single- and/ormulti-cysteine variant Cas9 protein described in Example 1, according tostandard procedures known to the ordinarily skilled artisan, tocovalently attach the acrydite-modified template nucleic acid to atleast one thiol group (e.g., a surface exposed thiol group) from thesingle- and/or multi-cysteine variant Cas9 protein described in Example1, to generate Cas9 fusion molecules (FIG. 6). Assays to test theefficiency of the Cas9 fusion molecule prepared as described in thisExample can be performed by analyzing the reaction products via anelectrophoretic mobility shift assay using standard procedures known tothe ordinary skilled artisan.

Example 6: Generation of Cas9 Fusion Molecules by Covalent Attachment ofa Cas9 Protein Molecule to Template Nucleic Acid Via EMCH and EDCCoupling Agents

To generate Cas9 fusion molecules, a N-[ε-Maleimidocaproic acid]hydrazide (EMCH)-modified Cas9 protein molecule is covalently attachedto a 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)-modifiedtemplate nucleic acid. The template nucleic acid is prepared such thatthe 5′-end of the template nucleic acid is modified to include acarboxyl moiety (i.e., —COOH), therefore resulting in a carboxy-modifiedtemplate nucleic acid. Alternatively, the template nucleic acid may bemodified such that the template nucleic acid includes a carboxyl moiety(i.e., —COOH) at its 3′-end or at an internal nucleic acid residue. Thecarboxy-modified template nucleic acid is then incubated with1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), according tostandard procedures known to the ordinarily skilled artisan, tocovalently attach EDC to the carboxy-modified template nucleic acid(FIG. 7), thereby forming a 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)-modified template nucleic acid.

The N-[ε-Maleimidocaproic acid] hydrazide (EMCH)-modified Cas9 proteinmolecule is prepared as described herein. The chemistry that couples theCas9 protein molecule to EMCH takes advantage of a surface-exposed thiolgroup on the Cas9 protein molecule (e.g., a cysteine residue) which iscapable of forming a covalent bond with EMCH. The Cas9 protein moleculemay be a wild-type Cas9 protein molecule. Alternatively, the Cas9protein molecule may be a single- or multi-cysteine variant Cas9 proteinmolecule as described in Example 1. Specifically, the Cas9 proteinmolecule is incubated in the presence of EMCH, according to standardprocedures known to the ordinarily skilled artisan, to form aN-[ε-Maleimidocaproic acid] hydrazide (EMCH)-modified Cas9 proteinmolecule.

The chemistry that couples the N-[ε-Maleimidocaproic acid] hydrazide(EMCH)-modified Cas9 protein molecule to the1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)-modified exogenousdonor template sequence takes advantage of the primary amine group ofthe N-[ε-Maleimidocaproic acid] hydrazide (EMCH) modified Cas9 proteinmolecule that can form a covalent bond with the1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)-modified templatenucleic acid. Specifically, a N-[ε-Maleimidocaproic acid] hydrazide(EMCH)-modified Cas9 protein molecule is incubated with the1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)-modified templatenucleic acid, according to standard procedures known to the ordinarilyskilled artisan, to generate a Cas9 fusion molecule (see also,Immunogenicity and protective efficacy of Bacillus anthracispoly-gamma-D-glutamic acid capsule covalently coupled to a proteincarrier using a novel triazine-based conjugation strategy. Joyce J, CookJ, Chabot D, Hepler R, Shoop W, Xu Q, Stambaugh T, Aste-Amezaga M, WangS, Indrawati L, Bruner M, Friedlander A, Keller P, Caulfield M J BiolChem 2006; (281):8 4831-4843). Assays to test the efficiency of the Cas9fusion molecule prepared as described in this Example can be performedby analyzing the reaction products via an electrophoretic mobility shiftassay using standard procedures known to the ordinary skilled artisan.

Example 7: Generation of Cas9 Fusion Molecules Via Non-Covalent CouplingAgents

To generate Cas9 fusion molecules, a Cas9 protein molecule, covalentlylinked to biotin, and a template nucleic acid, covalently linked tobiotin, are non-covalently attached via the interaction of the biotinmoiety of the Cas9 protein molecule and the biotin moiety of thetemplate nucleic acid each to a unique single monomer of tetramericstreptavidin. Using standard procedures known to the ordinarily skilledartisan, a Cas9 protein molecule is covalently attached to biotin via apolypeptide linker. The polypeptide linker may be one of the linkersdisclosed herein (e.g., an XTEN linker, a GGC3 linker, a GGS6 linker, ora GGS9 linker). Generally, the polypeptide linker is sufficiently longto allow the Cas9 protein molecule to interact with the template nucleicacid without steric interference. The template nucleic acid, which isalso covalently linked to a biotin molecule, is incubated with theCas9-Biotin protein molecule in the presence of streptavidin, accordingto standard procedures known to the ordinarily skilled artisan, therebyforming a Cas9 fusion molecule (FIG. 8). Assays to test the efficiencyof the Cas9 fusion molecule prepared as described in this Example can beperformed by analyzing the reaction products via an electrophoreticmobility shift assay using standard procedures known to the ordinaryskilled artisan.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned herein arehereby incorporated by reference in their entirety as if each individualpublication, patent or patent application was specifically andindividually indicated to be incorporated by reference. In case ofconflict, the present application, including any definitions herein,will control.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

REFERENCES

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1-64. (canceled)
 65. A Cas9 fusion molecule, comprising a Cas9 moleculecovalently linked to a template nucleic acid by a polypeptide linker.66. The Cas9 fusion molecule of claim 65, wherein the Cas9 moleculecomprises at least one surface exposed cysteine residue.
 67. The Cas9fusion molecule of claim 66, wherein (i) the Cas9 molecule comprises asurface exposed thiol group; (ii) the template nucleic acid comprises amaleimide modification; and/or (iii) the template nucleic acid comprisesan acrydite modification.
 68. The Cas9 fusion molecule of claim 65,wherein the Cas9 molecule comprisessuccinimidyl-6-hydrazino-nicotinamide (S-HyNic); or the Cas9 moleculecomprises a tag linked to the Cas9 molecule.
 69. The Cas9 fusionmolecule of claim 68, wherein the Cas9 molecule comprises a tag linkedto the Cas9 molecule, and the template nucleic acid comprises ahaloalkane, wherein the tag comprises a HaloTag molecule, wherein theHaloTag molecule is linked to the template nucleic acid using an SN₂reaction.
 70. The Cas9 fusion molecule of claim 65 wherein the templatenucleic acid comprises a double stranded nucleic acid or a singlestranded nucleic acid.
 71. The Cas9 fusion molecule of claim 65, whereinthe Cas9 molecule is selected from the group consisting of a wild-typeCas9 molecule, a Cas9 nickase molecule, a split Cas9 molecule, aninducible Cas9 molecule, an enzymatically active Cas9 (eaCas9) molecule,and an enzymatically inactive Cas9 (eiCas9) molecule.
 72. The Cas9fusion molecule of claim 65, wherein the polypeptide linker is between 3and 100 amino acids in length.
 73. The Cas9 fusion molecule of claim 65,wherein the polypeptide linker comprises an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 206-214.
 74. A gene editingsystem, comprising at least one Cas9 fusion molecule comprising a Cas9molecule covalently linked to a template nucleic acid by a polypeptidelinker, and at least one gRNA molecule.
 75. The gene editing system ofclaim 74, i) wherein the at least one gRNA molecule and the Cas9 fusionmolecule are designed to associate with a target nucleic acid andgenerate a double strand break on the target nucleic acid, wherein thedouble strand break is repaired by at least one DNA repair pathway,thereby producing a modified target nucleic acid; or ii) wherein theCas9 molecule is a Cas9 nickase molecule.
 76. A pharmaceuticalcomposition comprising the gene editing system of claim
 74. 77. A methodof modifying a target nucleic acid in a cell, the method comprising:contacting the cell with a gRNA molecule and the Cas9 fusion molecule ofclaim 65; wherein the gRNA molecule and the Cas9 fusion moleculeassociate with the target nucleic acid and generate a double strandbreak in the target nucleic acid; and wherein the double strand break isrepaired by gene correction using the template nucleic acid of the Cas9fusion molecule.
 78. A method of modifying a target nucleic acid in acell, the method comprising: contacting the cell with a first gRNAmolecule; a first Cas9 molecule; a second gRNA molecule; and a secondCas9 molecule; wherein at least one of the first and second Cas9molecule is covalently linked to a template nucleic acid by apolypeptide linker, wherein the first gRNA molecule and the first Cas9molecule associate with the target nucleic acid and generate a firstsingle strand cleavage event on a first strand of the target nucleicacid; wherein the second gRNA molecule and the second Cas9 moleculeassociate with the target nucleic acid and generate a second singlestrand cleavage event on a second strand of the target nucleic acid,thereby forming a double strand break having a first overhang and asecond overhang; and wherein the first overhang and the second overhangin the target nucleic acid are repaired by gene correction using thetemplate nucleic acid.
 79. The method of claim 78, wherein the firstCas9 molecule is covalently linked to the template nucleic acid by apolypeptide linker.
 80. The method of claim 78, wherein both the firstCas9 molecule and the second Cas9 molecule are covalently linked to thetemplate nucleic acid by a polypeptide linker.
 81. The method of claim78, wherein each Cas9 has N-terminal RuvC-like domain cleavage activitybut no HNH-like domain cleavage activity; or each Cas9 has HNH-likedomain cleavage activity but no N-terminal RuvC-like domain cleavageactivity.
 82. A gene editing system, comprising (i) a first Cas9 fusionmolecule, wherein the first Cas9 fusion molecule comprises a first Cas9nickase molecule covalently linked to a template nucleic acid by apolypeptide linker; a first gRNA molecule; a second Cas9 fusionmolecule, wherein the second Cas9 fusion molecule comprises a secondCas9 nickase molecule covalently linked to the template nucleic acid bya polypeptide linker; and a second gRNA molecule; or (ii) a first Cas9fusion molecule, wherein the first Cas9 fusion molecule comprises afirst Cas9 nickase molecule covalently linked to a first templatenucleic acid by a polypeptide linker; a first gRNA molecule; a secondCas9 fusion molecule, wherein the second Cas9 fusion molecule comprisesa second Cas9 nickase molecule covalently linked to a second templatenucleic acid by a polypeptide linker; and a second gRNA molecule. 83.The gene editing system of claim 82, wherein the first gRNA molecule andthe first Cas9 fusion molecule are designed to associate with a targetnucleic acid and generate a first single strand break on a first strandof the target nucleic acid; wherein the second gRNA molecule and thesecond Cas9 fusion molecule are designed to associate with the targetnucleic acid and generate a second single strand break on a secondstrand of the target nucleic acid, thereby forming a double strand breakin the target nucleic acid having a first overhang and a secondoverhang; and wherein the double strand break is repaired by at leastone DNA repair pathway, thereby producing a modified target nucleicacid.
 84. The gene editing system of claim 82, wherein each Cas9 nickasemolecule has N-terminal RuvC-like domain cleavage activity but noHNH-like domain cleavage activity; or each Cas9 nickase molecule hasHNH-like domain cleavage activity but no N-terminal RuvC-like domaincleavage activity.
 85. A nucleic acid molecule encoding the Cas9 fusionmolecule of claim
 1. 86. A vector, liposome or nanoparticle comprisingthe nucleic acid molecule of claim
 85. 87. The vector, liposome ornanoparticle of claim 86, wherein the vector is an AAV vector or alentiviral vector.